U.S. patent application number 11/286262 was filed with the patent office on 2006-06-29 for mass spectrometer.
This patent application is currently assigned to Micromass UK Limited. Invention is credited to Robert Harold Bateman.
Application Number | 20060138320 11/286262 |
Document ID | / |
Family ID | 27617664 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060138320 |
Kind Code |
A1 |
Bateman; Robert Harold |
June 29, 2006 |
Mass spectrometer
Abstract
A method of mass spectrometry is disclosed wherein a collision,
fragmentation or reaction device is repeatedly switched between a
high fragmentation or reaction mode and a low fragmentation or
reaction mode. Parent ions from a first sample are passed through
the device and parent ion mass spectra and fragmentation ion mass
spectra are obtained. Parent ions from a second sample are then
passed through the device and a second set of parent ion mass
spectra and fragmentation ion mass spectra are obtained. The mass
spectra are then compared and if either certain parent ions or
certain fragmentation ions in the two samples are expressed
differently then further analysis is performed to seek to identify
the ions which are expressed differently in the two different
samples.
Inventors: |
Bateman; Robert Harold;
(Cheshire, GB) |
Correspondence
Address: |
WATERS INVESTMENTS LIMITED;C/O WATERS CORPORATION
34 MAPLE STREET - LG
MILFORD
MA
01757
US
|
Assignee: |
Micromass UK Limited
Manchester
GB
|
Family ID: |
27617664 |
Appl. No.: |
11/286262 |
Filed: |
November 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10464513 |
Jun 19, 2003 |
6982414 |
|
|
11286262 |
Nov 23, 2005 |
|
|
|
60412800 |
Sep 24, 2002 |
|
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Current U.S.
Class: |
250/288 ;
250/281 |
Current CPC
Class: |
H01J 49/34 20130101;
H01J 49/0027 20130101; H01J 49/0045 20130101; H01J 49/0031
20130101; H01J 49/10 20130101 |
Class at
Publication: |
250/288 ;
250/281 |
International
Class: |
H01J 49/00 20060101
H01J049/00; B01D 59/44 20060101 B01D059/44 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 24, 2002 |
GB |
0217146 |
Aug 12, 2002 |
GB |
0218719 |
Sep 20, 2002 |
GB |
0221914 |
Mar 13, 2003 |
GB |
0305796 |
Claims
1. A method of mass spectrometry comprising: passing parent or
precursor ions from a first sample to a collision, fragmentation or
reaction device; repeatedly switching, altering or varying said
collision, fragmentation or reaction device between a first mode
wherein at least some of said parent or precursor ions from said
first sample are fragmented or reacted to form one or more
fragment, product, daughter or adduct ions and a second mode
wherein substantially fewer parent or precursor ions are fragmented
or reacted; passing parent or precursor ions from a second sample
to a collision, fragmentation or reaction device; repeatedly
switching, altering or varying said collision, fragmentation or
reaction device between a first mode wherein at least some of said
parent or precursor ions from said second sample are fragmented or
reacted to form one or more fragment, product, daughter or adduct
ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented or reacted; automatically determining
the intensity of first parent or precursor ions from said first
sample which have a first mass to charge ratio; automatically
determining the intensity of second parent or precursor ions from
said second sample which have said same first mass to charge ratio;
and comparing the intensity of said first parent or precursor ions
with the intensity of said second parent or precursor ions; wherein
if the intensity of said first parent or precursor ions differs
from the intensity of said second parent or precursor ions by more
than a predetermined amount then either said first parent or
precursor ions and/or said second parent or precursor ions are
considered to be parent or precursor ions of interest; and wherein
said collision, fragmentation or reaction device is selected from
the group consisting of: (i) a Surface Induced Dissociation ("SID")
fragmentation device; (ii) an Electron Transfer Dissociation
fragmentation device; (iii) an Electron Capture Dissociation
fragmentation device; (iv) an Electron Collision or Impact
Dissociation fragmentation device; (v) a Photo Induced Dissociation
("PID") fragmentation device; (vi) a Laser Induced Dissociation
fragmentation device; (vii) an infrared radiation induced
dissociation device; (viii) an ultraviolet radiation induced
dissociation device; (ix) a nozzle-skimmer interface fragmentation
device; (x) an in-source fragmentation device; (xi) an ion-source
Collision Induced Dissociation fragmentation device; (xii) a
thermal or temperature source fragmentation device; (xiii) an
electric field induced fragmentation device; (xiv) a magnetic field
induced fragmentation device; (xv) an enzyme digestion or enzyme
degradation fragmentation device; (xvi) an ion-ion reaction
fragmentation device; (xvii) an ion-molecule reaction fragmentation
device; (xviii) an ion-atom reaction fragmentation device; (xix) an
ion-metastable ion reaction fragmentation device; (xx) an
ion-metastable molecule reaction fragmentation device; (xxi) an
ion-metastable atom reaction fragmentation device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
2. A method of mass spectrometry comprising: passing parent or
precursor ions from a first sample to a collision, fragmentation or
reaction device; repeatedly switching, altering or varying said
collision, fragmentation or reaction device between a first mode
wherein at least some of said parent or precursor ions from said
first sample are fragmented or reacted to form one or more
fragment, product, daughter or adduct ions and a second mode
wherein substantially fewer parent or precursor ions are fragmented
or reacted; passing parent or precursor ions from a second sample
to a collision, fragmentation or reaction device; repeatedly
switching, altering or varying said collision, fragmentation or
reaction device between a first mode wherein at least some of said
parent or precursor ions from said second sample are fragmented or
reacted to form one or more fragment, product, daughter or adduct
ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented or reacted; automatically determining
the intensity of first parent or precursor ions from said first
sample which have a first mass to charge ratio; automatically
determining the intensity of second parent or precursor ions from
said second sample which have said same first mass to charge ratio;
determining a first ratio of the intensity of said first parent or
precursor ions to the intensity of other parent or precursor ions
in said first sample; determining a second ratio of the intensity
of said second parent or precursor ions to the intensity of other
parent or precursor ions in said second sample; and comparing said
first ratio with said second ratio; wherein if said first ratio
differs from said second ratio by more than a predetermined amount
then either said first parent or precursor ions and/or said second
parent or precursor ions are considered to be parent or precursor
ions of interest; and wherein said collision, fragmentation or
reaction device is selected from the group consisting of: (i) a
Surface Induced Dissociation ("SID") fragmentation device; (ii) an
Electron Transfer Dissociation fragmentation device; (iii) an
Electron Capture Dissociation fragmentation device; (iv) an
Electron Collision or Impact Dissociation fragmentation device; (v)
a Photo Induced Dissociation ("PID") fragmentation device; (vi) a
Laser Induced Dissociation fragmentation device; (vii) an infrared
radiation induced dissociation device; (viii) an ultraviolet
radiation induced dissociation device; (ix) a nozzle-skimmer
interface fragmentation device; (x) an in-source fragmentation
device; (xi) an ion-source Collision Induced Dissociation
fragmentation device; (xii) a thermal or temperature source
fragmentation device; (xiii) an electric field induced
fragmentation device; (xiv) a magnetic field induced fragmentation
device; (xv) an enzyme digestion or enzyme degradation
fragmentation device; (xvi) an ion-ion reaction fragmentation
device; (xvii) an ion-molecule reaction fragmentation device;
(xviii) an ion-atom reaction fragmentation device; (xix) an
ion-metastable ion reaction fragmentation device; (xx) an
ion-metastable molecule reaction fragmentation device; (xxi) an
ion-metastable atom reaction fragmentation device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
3. A method as claimed in claim 2, wherein either said other parent
or precursor ions present in said first sample and/or said other
parent or precursor ions present in said second sample are either
endogenous or exogenous to said sample.
4. (canceled)
5. A method as claimed in claims 2, wherein said other parent or
precursor ions present in said first sample and/or said other
parent or precursor ions present in said second sample are
additionally used as a chromatographic retention time standard.
6. A method as claimed in claim 1, wherein said predetermined
amount is selected from the group consisting of: (i) 1%; (ii) 10%;
(iii) 50%; (iv) 100%; (v) 150%; (vi) 200%; (vii) 250%; (viii) 300%;
(ix) 350%; (x) 400%; (xi) 450%; (xii) 500%; (xiii) 1000%; (xiv)
5000%; or (xv) 10000%.
7-13. (canceled)
14. A method as claimed in claim 1, further comprising the step of
identifying said parent or precursor ions of interest.
15. A method as claimed in claim 14, wherein the step of
identifying said parent or precursor ions of interest comprises
either determining the mass to charge ratio of said parent or
precursor ions of interest or identifying one or more fragment,
product, daughter or adduct ions which are determined to result
from fragmentation or reaction of said parent or precursor ions of
interest.
16. A method as claimed in claim 15, wherein the mass to charge
ratio of said parent or precursor ions of interest or said one or
more fragment, product, daughter or adduct ions is determined to
less than or equal to 20 ppm, 15 ppm, 10 ppm or 5 ppm.
17. A method as claimed in claim 15, further comprising comparing
the determined mass to charge ratio of said parent or precursor
ions of interest with a database of ions and their corresponding
mass to charge ratios.
18. (canceled)
19. (canceled)
20. A method as claimed in claim 4815, wherein the step of
identifying parent or precursor ions of interest comprises
determining whether said parent or precursor ions of interest are
observed in a mass spectrum obtained when said collision,
fragmentation or reaction device is in said second mode for a
certain time period and said fragment, product, daughter or adduct
ions are observed in a mass spectrum obtained either immediately
before said certain time period, when said collision, fragmentation
or reaction device is in said first mode, or immediately after said
certain time period, when said collision, fragmentation or reaction
device is in said first mode.
21. (canceled)
22. A method as claimed in claim 15, wherein the step of
identifying said parent or precursor ions of interest comprises
comparing the elution time or profile of said parent or precursor
ions of interest with the pseudo-elution time or profile of said
fragment, product, daughter or adduct ions.
23. A method of mass spectrometry as claimed in claim 1, further
comprising determining that ions are parent or precursor ions by
comparing two mass spectra obtained one after the other, a first
mass spectrum being obtained when said collision, fragmentation or
reaction device was in said first mode and a second mass spectrum
being obtained when said collision, fragmentation or reaction
device was in said second mode, wherein ions are determined to be
parent or precursor ions if a peak corresponding to said ions in
said second mass spectrum is more intense than a peak corresponding
to said ions in said first mass spectrum.
24. A method as claimed in claim 1, further comprising determining
that ions are determined to be fragment, product, daughter or
adduct ions by comparing two mass spectra obtained one after the
other, a first mass spectrum being obtained when said collision,
fragmentation or reaction device was in said first mode and a
second mass spectrum being obtained when said collision,
fragmentation or reaction device was in said second mode, wherein
ions are determined to be fragment, product, daughter or adduct
ions if a peak corresponding to said ions in said first mass
spectrum is more intense than a peak corresponding to said ions in
said second mass spectrum.
25. A method as claimed in claim 1, further comprising: providing a
mass filter upstream of said collision, fragmentation or reaction
device wherein said mass filter is arranged to transmit ions having
mass to charge ratios within a first range but to substantially
attenuate ions having mass to charge ratios within a second range;
and wherein ions are determined to be fragment, product, daughter
or adduct ions if they are determined to have a mass to charge
ratio falling within said second range.
26. A method as claimed in claim 1, wherein said first parent or
precursor ions and said second parent or precursor ions are
determined to have mass to charge ratios which differ by less than
or equal to 40 ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 10 ppm
or 5 ppm.
27. A method as claimed in claim 1, wherein said first parent or
precursor ions and said second parent or precursor ions are
determined to have eluted from a chromatography column after
substantially the same elution time.
28. A method as claimed in claim 1, wherein said first parent or
precursor ions are determined to give rise to one or more first
fragment, product, daughter or adduct ions and said second parent
or precursor ions are determined to give rise to one or more second
fragment, product, daughter or adduct ions, wherein said one or
more first fragment, product, daughter or adduct ions and said one
or more second fragment, product, daughter or adduct ions have
substantially the same mass to charge ratio.
29. A method as claimed in claim 28, wherein the mass to charge
ratio of said one or more first fragment, product, daughter or
adduct ions and said one or more second fragment, product, daughter
or adduct ions are determined to differ by less than or equal to 40
ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 10 ppm or 5 ppm.
30. A method as claimed in claim 1, wherein said first parent or
precursor ions are determined to give rise to one or more first
fragment, product, daughter or adduct ions and said second parent
or precursor ions are determined to give rise to one or more second
fragment, product, daughter or adduct ions and wherein said first
parent or precursor ions and said second parent or precursor ions
are observed in mass spectra relating to data obtained in said
second mode at a certain point in time and said one or more first
and second fragment, product, daughter or adduct ions are observed
in mass spectra relating to data obtained either immediately before
said certain point in time when said collision, fragmentation or
reaction device is in said first mode or immediately after said
certain point in time when said collision, fragmentation or
reaction device is in said first mode.
31. A method as claimed in claim 1, wherein said first parent or
precursor ions are determined to give rise to one or more first
fragment, product, daughter or adduct ions and said second parent
or precursor ions are determined to give rise to one or more second
fragment, product, daughter or adduct ions and wherein said first
fragment, product, daughter or adduct ions have substantially the
same pseudo-elution time as said second fragment, product, daughter
or adduct ions.
32. A method as claimed in claim 1, wherein said first parent or
precursor ions are determined to give rise to one or more first
fragment, product, daughter or adduct ions and said second parent
or precursor ions are determined to give rise to one or more second
fragment, product, daughter or adduct ions and wherein said first
parent or precursor ions are determined to have an elution profile
which correlates with a pseudo-elution profile of said first
fragment, product, daughter or adduct ions and wherein said second
parent or precursor ions are determined to have an elution profile
which correlates with a pseudo-elution profile of said second
fragment, product, daughter or adduct ions.
33. A method as claimed in claim 1, wherein said first parent or
precursor ions and said second parent or precursor ions are
determined to be either multiply charged or to have the same charge
state.
34. (canceled)
35. A method as claimed in claim 1, wherein fragment, product,
daughter or adduct ions which are determined to result from the
fragmentation or reaction of said first parent or precursor ions
are determined to have the same charge state as fragment, product,
daughter or adduct ions which are determined to result from the
fragmentation or reaction of said second parent or precursor
ions.
36. A method as claimed in claim 1, wherein said first sample
and/or said second sample comprise a plurality of different
biopolymers, proteins, peptides, polypeptides, oligionucleotides,
oligionucleosides, amino acids, carbohydrates, sugars, lipids,
fatty acids, vitamins, hormones, portions or fragments of DNA,
portions or fragments of cDNA, portions or fragments of RNA,
portions or fragments of mRNA, portions or fragments of tRNA,
polyclonal antibodies, monoclonal antibodies, ribonucleases,
enzymes, metabolites, polysaccharides, phosphorylated peptides,
phosphorylated proteins, glycopeptides, glycoproteins or
steroids.
37. A method as claimed in claim 1, wherein said first sample
and/or said second sample comprise at least 2, 5, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 molecules
having different identities.
38. A method as claimed in claim 1, wherein either: (i) said first
sample is taken from a diseased organism and said second sample is
taken from a non-diseased organism; (ii) said first sample is taken
from a treated organism and said second sample is taken from a
non-treated organism; or (iii) said first sample is taken from a
mutant organism and said second sample is taken from a wild type
organism.
39. A method as claimed in claim 1, wherein molecules from said
first and/or second samples are separated from a mixture of other
molecules prior to being ionised by: (i) High Performance Liquid
Chromatography ("HPLC"); (ii) anion exchange; (iii) anion exchange
chromatography; (iv) cation exchange; (v) cation exchange
chromatography; (vi) ion pair reversed-phase chromatography; (vii)
chromatography; (viii) single dimensional electrophoresis; (ix)
multi-dimensional electrophoresis; (x) size exclusion; (xi)
affinity; (xii) reverse phase chromatography; (xiii) Capillary
Electrophoresis Chromatography ("CEC"); (xiv) electrophoresis; (xv)
ion mobility separation; (xvi) Field Asymmetric Ion Mobility
Separation ("FAIMS"); or (xvi) capillary electrophoresis.
40. A method as claimed in claim 1, wherein said first and second
sample ions comprise peptide ions.
41. A method as claimed in claim 40, wherein said peptide ions
comprise the digest products of one or more proteins.
42. A method as claimed in claim 40, further comprising the step of
attempting to identify a protein which correlates with said parent
or precursor ions of interest.
43. A method as claimed in claim 42, further comprising either: (i)
determining which peptide products are predicted to be formed when
a protein is digested and determining whether any predicted peptide
product(s) correlate with parent or precursor ions of interest or
(ii) determining whether said parent or precursor ions of interest
correlate with one or more proteins.
44. (canceled)
45. A method as claimed in claim 1, wherein said first and second
samples are taken either from the same organism or from different
organisms.
46. (canceled)
47. A method as claimed in claim 1, further comprising the step of
confirming that said first parent or precursor ions and/or said
second parent or precursor ions are not fragment, product, daughter
or adduct ions caused by fragmentation of parent or precursor ions
in said collision, fragmentation or reaction device.
48. A method as claimed in claim 47, further comprising: comparing
a first mass spectrum relating to data obtained in said first mode
with a second mass spectrum relating to data obtained in said
second mode, said mass spectra being obtained at substantially the
same time; and determining that said first and/or said second
parent or precursor ions are not fragment, product, daughter or
adduct ions if said first and/or said second parent or precursor
ions have a greater intensity in the second mass spectrum relative
to the first mass spectrum.
49. (canceled)
50. (canceled)
51. A mass spectrometer comprising: a collision, fragmentation or
reaction device which is arranged and adapted to be repeatedly
switched, altered or varied in use between a first mode wherein at
least some parent or precursor ions are fragmented or reacted into
one or more fragment, product, daughter or adduct ions and a second
mode wherein substantially fewer parent or precursor ions are
fragmented or reacted; a mass analyser; and a control system which
in use: (i) determines the intensity of first parent or precursor
ions from a first sample which have a first mass to charge ratio;
(ii) determines the intensity of second parent or precursor ions
from a second sample which have said same first mass to charge
ratio; and (iii) compares the intensity of said first parent or
precursor ions with the intensity of said second parent or
precursor ions; wherein if the intensity of said first parent or
precursor ions differs from the intensity of said second parent or
precursor ions by more than a predetermined amount then either said
first parent or precursor ions and/or said second parent or
precursor ions are considered to be parent or precursor ions of
interest; wherein said collision, fragmentation or reaction device
is selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") fragmentation device; (ii) an Electron
Transfer Dissociation fragmentation device; (iii) an Electron
Capture Dissociation fragmentation device; (iv) an Electron
Collision or Impact Dissociation fragmentation device; (v) a Photo
Induced Dissociation ("PID") fragmentation device; (vi) a Laser
Induced Dissociation fragmentation device; (vii) an infrared
radiation induced dissociation device; (viii) an ultraviolet
radiation induced dissociation device; (ix) a nozzle-skimmer
interface fragmentation device; (x) an in-source fragmentation
device; (xi) an ion-source Collision Induced Dissociation
fragmentation device; (xii) a thermal or temperature source
fragmentation device; (xiii) an electric field induced
fragmentation device; (xiv) a magnetic field induced fragmentation
device; (xv) an enzyme digestion or enzyme degradation
fragmentation device; (xvi) an ion-ion reaction fragmentation
device; (xvii) an ion-molecule reaction fragmentation device;
(xviii) an ion-atom reaction fragmentation device; (xix) an
ion-metastable ion reaction fragmentation device; (xx) an
ion-metastable molecule reaction fragmentation device; (xxi) an
ion-metastable atom reaction fragmentation device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
52. A mass spectrometer comprising: a collision, fragmentation or
reaction device which is arranged and adapted to be repeatedly
switched, altered or varied in use between a first mode wherein at
least some parent or precursor ions are fragmented or reacted into
one or more fragment, product, daughter or adduct ions and a second
mode wherein substantially fewer parent or precursor ions are
fragmented or reacted; a mass analyser; and a control system which
in use: (i) determines the intensity of first parent or precursor
ions from a first sample which have a first mass to charge ratio;
(ii) determines the intensity of second parent or precursor ions
from a second sample which have said same first mass to charge
ratio; (iii) determines a first ratio of the intensity of said
first parent or precursor ions to the intensity of other parent or
precursor ions in said first sample; (iv) determines a second ratio
of the intensity of said second parent or precursor ions to the
intensity of other parent or precursor ions in said second sample;
and (v) compares said first ratio with said second ratio; wherein
if said first ratio differs from said second ratio by more than a
predetermined amount then either said first parent or precursor
ions and/or said second parent or precursor ions are considered to
be parent or precursor ions of interest; wherein said collision,
fragmentation or reaction device is selected from the group
consisting of: (i) a Surface Induced Dissociation ("SID")
fragmentation device; (ii) an Electron Transfer Dissociation
fragmentation device; (iii) an Electron Capture Dissociation
fragmentation device; (iv) an Electron Collision or Impact
Dissociation fragmentation device; (v) a Photo Induced Dissociation
("PID") fragmentation device; (vi) a Laser Induced Dissociation
fragmentation device; (vii) an infrared radiation induced
dissociation device; (viii) an ultraviolet radiation induced
dissociation device; (ix) a nozzle-skimmer interface fragmentation
device; (x) an in-source fragmentation device; (xi) an ion-source
Collision Induced Dissociation fragmentation device; (xii) a
thermal or temperature source fragmentation device; (xiii) an
electric field induced fragmentation device; (xiv) a magnetic field
induced fragmentation device; (xv) an enzyme digestion or enzyme
degradation fragmentation device; (xvi) an ion-ion reaction
fragmentation device; (xvii) an ion-molecule reaction fragmentation
device; (xviii) an ion-atom reaction fragmentation device; (xix) an
ion-metastable ion reaction fragmentation device; (xx) an
ion-metastable molecule reaction fragmentation device; (xxi) an
ion-metastable atom reaction fragmentation device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
53. A mass spectrometer as claimed in claim 51, further comprising
an ion source.
54. A mass spectrometer as claimed in claim 53, wherein said ion
source is selected from the group consisting of: (i) an
Electrospray ionisation ("ESI") ion source; (ii) an Atmospheric
Pressure Photo Ionisation ("APPI") ion source; (iii) an Atmospheric
Pressure Chemical Ionisation ("APCI") ion source; (iv) a Matrix
Assisted Laser Desorption Ionisation ("MALDI") ion source; (v) a
Laser Desorption Ionisation ("LDI") ion source; (vi) an Atmospheric
Pressure Ionisation ("API") ion source; (vii) a Desorption
Ionisation on Silicon ("DIOS") ion source; (viii) an Electron
Impact ("El") ion source; (ix) a Chemical Ionisation ("Cl") ion
source; (x) a Field Ionisation ("FI") ion source; (xi) a Field
Desorption ("FD") ion source; (xii) an Inductively Coupled Plasma
("ICP") ion source; (xiii) a Fast Atom Bombardment ("FAB") ion
source; (xiv) a Liquid Secondary Ion Mass Spectrometry ("LSIMS")
ion source; (xv) a Desorption Electrospray Ionisation ("DESI") ion
source; (xvi) a Nickel-63 radioactive ion source; (xvii) an
Atmospheric Pressure Matrix Assisted Laser Desorption Ionisation
ion source; and (xviii) a Thermospray ion source.
55. A mass spectrometer as claimed in claim 53, wherein said ion
source comprises a pulsed or continuous ion source.
56. A mass spectrometer as claimed in claim 51, wherein said mass
analyser is selected from the group consisting of: (i) a Time of
Flight mass analyser; (ii) an orthogonal acceleration Time of
Flight mass analyser; (iii) an axial acceleration Time of Flight
mass analyser; (iv) a quadrupole mass analyser; (v) a 2D or linear
quadrupole ion trap mass analyser; (vi) a Paul or 3D quadrupole ion
trap mass analyser; (vii) a Penning trap mass analyser; (viii) an
ion trap mass analyser; (ix) a magnetic sector mass analyser; (x)
Ion Cyclotron Resonance ("ICR") mass analyser; (xi) a Fourier
Transform Ion Cyclotron Resonance ("FTICR") mass analyser; (xii) an
electrostatic or orbitrap mass analyser; (xiii) a Fourier Transform
electrostatic or orbitrap mass analyser; and (xiv) a Fourier
Transform mass analyser.
57. A mass spectrometer as claimed in claim 53, wherein said ion
source is provided with an eluent over a period of time, said
eluent having been separated from a mixture by means of liquid
chromatography gas chromatography or capillary electrophoresis.
58-69. (canceled)
70. A mass spectrometer as claimed in claim 51, wherein molecules
from said first and/or second samples are separated from a mixture
of other molecules prior to being ionised by: (i) High Performance
Liquid Chromatography ("HPLC"); (ii) anion exchange; (iii) anion
exchange chromatography; (iv) cation exchange; (v) cation exchange
chromatography; (vi) ion pair reversed-phase chromatography; (vii)
chromatography; (viii) single dimensional electrophoresis; (ix)
multi-dimensional electrophoresis; (x) size exclusion; (xi)
affinity; (xii) reverse phase chromatography; (xiii) Capillary
Electrophoresis Chromatography ("CEC"); (xiv) electrophoresis; (xv)
ion mobility separation; (xvi) Field Asymmetric Ion Mobility
Separation ("FAIMS"); or (xvi) capillary electrophoresis.
71-74. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of U.S. patent appliction
Ser. No. 10/464,513 filed 19 Jun. 2003, which claims priority from
UK patent application no. 0217146.0 filed 24 Jul. 2002, UK patent
application no. 0218719.3 filed 12 Aug. 2002, UK patent application
no. 0221914.5 filed 20 Sep. 2002, UK patent application no.
0305796.5 filed 13 Mar. 2003 and US provisional patent application
Ser. No. 60/412,800 filed 24 Sep. 2002. The contents of these
applications are incorporated herein by reference.
FIELD OF INVENTION
[0002] The present invention relates to a method of mass
spectrometry and a mass spectrometer.
BACKGROUND OF INVENTION
[0003] It has become common practice to analyse proteins by first
enzymatically or chemically digesting the protein and then
analysing the peptide products by mass spectrometry. The mass
spectrometry analysis of the peptide products normally entails
measuring the mass of the peptide products. This method is
sometimes referred to as "peptide mapping" or "peptide
fingerprinting".
[0004] It is also known to induce parent or precursor peptide ions
to fragment and to then measure the mass of one or more fragment or
daughter ions as a way of seeking to identify the parent or
precursor peptide ion. The fragmentation pattern of a peptide ion
has also been shown to be a successful way of distinguishing
isobaric peptide ions. Thus the mass to charge ratio of one or more
fragment or daughter ions may be used to identify the parent or
precursor peptide ion and hence the protein from which the peptide
was derived. In some instances the partial sequence of the peptide
can also be determined from the fragment or daughter ion spectrum.
This information may be used to determine candidate proteins by
searching protein and genomic databases.
[0005] Alternatively, a candidate protein may be eliminated or
confirmed by comparing the masses of one or more observed fragment
or daughter ions with the masses of fragment or daughter ions which
might be expected to be observed based upon the peptide sequence of
the candidate protein in question. The confidence in the
identification increases as more peptide parent or precursor ions
are induced to fragment and their fragment masses are shown to
match those expected.
SUMMARY OF INVENTION
[0006] According to an aspect of the present invention there is
provided a method of mass spectrometry comprising:
[0007] passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device;
[0008] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the first sample
are fragmented or reacted to form one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0009] passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device; and
[0010] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the second sample
are fragmented or reacted to form one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0011] automatically determining the intensity of first parent or
precursor ions from the first sample which have a first mass to
charge ratio;
[0012] automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first
mass to charge ratio; and
[0013] comparing the intensity of the first parent or precursor
ions with the intensity of the second parent or precursor ions;
[0014] wherein if the intensity of the first parent or precursor
ions differs from the intensity of the second parent or precursor
ions by more than a predetermined amount then either the first
parent or precursor ions and/or the second parent or precursor ions
are considered to be parent or precursor ions of interest; and
[0015] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0016] According to another aspect of the present invention there
is provided a method of mass spectrometry comprising:
[0017] passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device;
[0018] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the first sample
are fragmented or reacted to form one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0019] passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device; and
[0020] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the second sample
are fragmented or reacted to form one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0021] automatically determining the intensity of first parent or
precursor ions from the first sample which have a first mass to
charge ratio;
[0022] automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first
mass to charge ratio;
[0023] determining a first ratio of the intensity of the first
parent or precursor ions to the intensity of other parent or
precursor ions in the first sample;
[0024] determining a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
[0025] comparing the first ratio with the second ratio;
[0026] wherein if the first ratio differs from the second ratio by
more than a predetermined amount then either the first parent or
precursor ions and/or the second parent or precursor ions are
considered to be parent or precursor ions of interest; and
[0027] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0028] A reaction device should be understood as comprising a
device wherein ions, atoms or molecules are rearranged or reacted
so as to form a new species of ion, atom or molecule. An X--Y
reaction fragmentation device should be understood as meaning a
device wherein X and Y combine to form a product which then
fragments. This is different to a collision, fragmentation or
reaction device per se wherein ions may be caused to fragment
without first forming a product. An X--Y reaction device should be
understood as meaning a device wherein X and Y combine to form a
product which does not necessarily then fragment.
[0029] Other arrangements are also contemplated wherein instead of
determining a first ratio of first parent or precursor ions to
other parent or precursor ions, a first ratio of first parent or
precursor ions to certain fragment, product, daughter or adduct
ions may be determined. Similarly, a second ratio of second parent
or precursor ions to certain fragment, product, daughter or adduct
ions may be determined and the first and second ratios
compared.
[0030] The other parent or precursor ions present in the first
sample and/or the other parent or precursor ions present in the
second sample may either be endogenous or exogenous to the sample.
The other parent or precursor ions present in the first sample
and/or the other parent or precursor ions present in the second
sample may additionally be used as a chromatographic retention time
standard.
[0031] According to one embodiment parent or precursor ions,
preferably peptide ions, from two different samples are analysed in
separate experimental runs. In each experimental run parent or
precursor ions are passed to a collision, fragmentation or reaction
device. The collision, fragmentation or reaction device is
preferably repeatedly switched between a fragmentation or reaction
mode and a substantially non-fragmentation or reaction mode. The
ions emerging from the collision, fragmentation or reaction device
or which have been transmitted through the collision, fragmentation
or reaction device are then preferably mass analysed. The intensity
of parent or precursor ions having a certain mass to charge ratio
in one sample are then compared with the intensity of parent or
precursor ions having the same certain mass to charge ratio in the
other sample. A direct comparison of the parent or precursor ion
expression level may be made or the intensity of parent or
precursor ions in a sample may first be compared with an internal
standard. An indirect comparison may therefore be made between the
ratio of parent or precursor ions in one sample relative to the
intensity of parent or precursor ions relating to an internal
standard and the ratio of parent or precursor ions in the other
sample relative to the intensity of parent or precursor ions
relating to preferably the same internal standard. A comparison of
the two ratios may then be made. Although the preferred embodiment
is described as relating to comparing the parent or precursor ion
expression level in two samples, it is apparent that the expression
level of parent or precursor ions in three or more samples may be
compared.
[0032] Parent or precursor ions may be considered to be expressed
significantly differently in two samples if their expression level
differs by more than 1%, 10%, 50%, 100%, 150%, 200%, 250%, 300%,
350%, 400%, 450%, 500%, 1000%, 5000% or 10000%.
[0033] In the high fragmentation or reaction mode the collision,
fragmentation or reaction device may be supplied with a voltage
greater than or equal to 15V, 20V, 25V, 30V, 50V, 100V, 150V or
200V. Similarly, in the low fragmentation or reaction mode the
collision, fragmentation or reaction device may be supplied with a
voltage less than or equal to 5V, 4.5V, 4V, 3.5V, 3V, 2.5V, 2V,
1.5V, 1V, 0.5V or substantially OV. However, according to less
preferred embodiments, voltages below 15V may be supplied in the
first mode and/or voltages above 5V may be supplied in the second
mode. For example, in either the first or the second mode a voltage
of around 1OV may be supplied. Preferably, the voltage difference
between the two modes is at least 5V, 10V, 15V, 20V, 25V, 30V, 35V,
40V, 50V or more than 50V.
[0034] According to an embodiment in the high fragmentation or
reaction mode at least 50% of the ions entering the collision,
fragmentation or reaction device are arranged to have an energy
greater than or equal to 10 eV for a singly charged ion or an
energy greater than or equal to 20 eV for a doubly charged ion. The
collision, fragmentation or reaction device is preferably
maintained at a pressure selected from the group consisting of: (i)
greater than or equal to 0.0001 mbar; (ii) greater than or equal to
0.001 mbar; (iii) greater than or equal to 0.005 mbar; (iv) greater
than or equal to 0.01 mbar; (v) between 0.0001 and 100 mbar; and
(vi) between 0.001 and 10 mbar. Preferably, the collision,
fragmentation or reaction device is maintained at a pressure
selected from the group consisting of: (i) greater than or equal to
0.0001 mbar; (ii) greater than or equal to 0.0005 mbar; (iii)
greater than or equal to 0.001 mbar; (iv) greater than or equal to
0.005 mbar; (v) greater than or equal to 0.01 mbar; (vi) greater
than or equal to 0.05 mbar; (vii) greater than or equal to 0.1
mbar; (viii) greater than or equal to 0.5 mbar; (ix) greater than
or equal to 1 mbar; (x) greater than or equal to 5 mbar; and (xi)
greater than or equal to 10 mbar. Preferably, the collision,
fragmentation or reaction device is maintained at a pressure
selected from the group consisting of: (i) less than or equal to 10
mbar; (ii) less than or equal to 5 mbar; (iii) less than or equal
to 1 mbar; (iv) less than or equal to 0.5 mbar; (v) less than or
equal to 0.1 mbar; (vi) less than or equal to 0.05 mbar; (vii) less
than or equal to 0.01 mbar; (viii) less than or equal to 0.005
mbar; (ix) less than or equal to 0.001 mbar; (x) less than or equal
to 0.0005 mbar; and (xi) less than or equal to 0.0001 mbar.
[0035] According to a less preferred embodiment, gas in the
collision, fragmentation or reaction device may be maintained at a
first pressure when the collision, fragmentation or reaction device
is in the high fragmentation or reaction mode and at a second lower
pressure when the collision, fragmentation or reaction device is in
the low fragmentation or reaction mode. According to another less
preferred embodiment, gas in the collision, fragmentation or
reaction device may comprise a first gas or a first mixture of
gases when the collision, fragmentation or reaction device is in
the high fragmentation or reaction mode and a second different gas
or a second different mixture of gases when the collision,
fragmentation or reaction device is in the low fragmentation or
reaction mode.
[0036] Parent or precursor ions which are considered to be parent
or precursor ions of interest are preferably identified. This may
comprise determining the mass to charge ratio of the parent or
precursor ions of interest, preferably accurately to less than or
equal to 20 ppm, 15 ppm, 10 ppm or 5 ppm. The determined mass to
charge ratio of the parent or precursor ions of interest may then
be compared with a database of ions and their corresponding mass to
charge ratios and hence the identity of the parent or precursor
ions of interest can be established.
[0037] According to the preferred embodiment the step of
identifying the parent or precursor ions of interest comprises
identifying one or more fragment, product, daughter or adduct ions
which are determined to result from fragmentation of the parent or
precursor ions of interest. Preferably, the step of identifying one
or more fragment, product, daughter or adduct ions further
comprises determining the mass to charge ratio of the one or more
fragment, product, daughter or adduct ions to less than or equal to
20 ppm, 15 ppm, 10 ppm or 5 ppm.
[0038] The step of identifying first parent or precursor ions of
interest may comprise determining whether parent or precursor ions
are observed in a mass spectrum obtained when the collision,
fragmentation or reaction device is in the low fragmentation or
reaction mode for a certain time period and the first fragment,
product, daughter or adduct ions are observed in a mass spectrum
obtained either immediately before the certain time period, when
the collision, fragmentation or reaction device is in the high
fragmentation or reaction mode, or immediately after the certain
time period, when the collision, fragmentation or reaction device
is in the high fragmentation or reaction mode.
[0039] The step of identifying first parent or precursor ions of
interest may comprise comparing the elution times of parent or
precursor ions with the pseudo-elution time of first fragment,
product, daughter or adduct ions. The fragment, product, daughter
or adduct ions are referred to as having a pseudo-elution time
since fragment, product, daughter or adduct ions do not actually
physically elute from a chromatography column. However, since at
least some of the fragment, product, daughter or adduct ions are
fairly unique to particular parent or precursor ions, and the
parent or precursor ions may elute from the chromatography column
only at particular times, then the corresponding fragment, product,
daughter or adduct ions may similarly only be observed at
substantially the same elution time as their related parent or
precursor ions. Similarly, the step of identifying first parent or
precursor ions of interest may comprise comparing the elution
profiles of parent or precursor ions with the pseudo-elution
profile of first fragment, product, daughter or adduct ions. Again,
although fragment, product, daughter or adduct ions do not actually
physically elute from a chromatography column, they can be
considered to have an effective elution profile since they will
tend to be observed only when specific parent or precursor ions
elute from the column and as the intensity of the eluting parent or
precursor ions varies over a few seconds so similarly the intensity
of characteristic fragment, product, daughter or adduct ions will
also vary in a similar manner.
[0040] Ions may be determined to be parent or precursor ions by
comparing two mass spectra obtained one after the other, a first
mass spectrum being obtained when the collision, fragmentation or
reaction device was in a high fragmentation or reaction mode and a
second mass spectrum obtained when the collision, fragmentation or
reaction device was in a low fragmentation or reaction mode,
wherein ions are determined to be parent or precursor ions if a
peak corresponding to the ions in the second mass spectrum is more
intense than a peak corresponding to the ions in the first mass
spectrum. Similarly, ions may be determined to be fragment,
product, daughter or adduct ions if a peak corresponding to the
ions in the first mass spectrum is more intense than a peak
corresponding to the ions in the second mass spectrum. According to
another embodiment, a mass filter may be provided upstream of the
collision, fragmentation or reaction device wherein the mass filter
is arranged to transmit ions having mass to charge ratios within a
first range but to substantially attenuate ions having mass to
charge ratios within a second range and wherein ions are determined
to be fragment, product, daughter or adduct ions if they are
determined to have a mass to charge ratio falling within the second
range.
[0041] The first parent or precursor ions and the second parent or
precursor ions are preferably determined to have mass to charge
ratios which differ by less than or equal to 40 ppm, 35 ppm, 30
ppm, 25 ppm, 20 ppm, 15 ppm, 10 ppm or 5 ppm. The first parent or
precursor ions and the second parent or precursor ions may have
been determined to have eluted from a chromatography column after
substantially the same elution time. The first parent or precursor
ions may also have been determined to have given rise to one or
more first fragment, product, daughter or adduct ions and the
second parent or precursor ions may have been determined to have
given rise to one or more second fragment, product, daughter or
adduct ions, wherein the one or more first fragment, product,
daughter or adduct ions and the one or more second fragment,
product, daughter or adduct ions have substantially the same mass
to charge ratio. The mass to charge ratio of the one or more first
fragment, product, daughter or adduct ions and the one or more
second fragment, product, daughter or adduct ions may be determined
to differ by less than or equal to 40 ppm, 35 ppm, 30 ppm, 25 ppm,
20 ppm, 15 ppm, 10 ppm or 5 ppm.
[0042] The first parent or precursor ions may also be determined to
have given rise to one or more first fragment, product, daughter or
adduct ions and the second parent or precursor ions may have been
determined to have given rise to one or more second fragment,
product, daughter or adduct ions and wherein the first parent or
precursor ions and the second parent or precursor ions are observed
in mass spectra relating to data obtained in the low fragmentation
or reaction mode at a certain point in time and the one or more
first and second fragment, product, daughter or adduct ions are
observed in mass spectra relating to data obtained either
immediately before the certain point in time, when the collision,
fragmentation or reaction device is in the high fragmentation or
reaction mode, or immediately after the certain point in time, when
the collision, fragmentation or reaction device is in the high
fragmentation or reaction mode.
[0043] The first parent or precursor ions may be determined to have
given rise to one or more first fragment, product, daughter or
adduct ions and the second parent or precursor ions may be
determined to have given rise to one or more second fragment,
product, daughter or adduct ions if the first fragment, product,
daughter or adduct ions have substantially the same pseudo-elution
time as the second fragment, product, daughter or adduct ions.
[0044] The first parent or precursor ions may be determined to have
given rise to one or more first fragment, product, daughter or
adduct ions and the second parent or precursor ions may be
determined to have given rise to one or more second fragment,
product, daughter or adduct ions and wherein the first parent or
precursor ions are determined to have an elution profile which
correlates with a pseudo-elution profile of a first fragment,
product, daughter or adduct ion and wherein the corresponding
second parent or precursor ions are determined to have an elution
profile which correlates with a pseudo-elution profile of a second
fragment, product, daughter or adduct ion.
[0045] According to another embodiment the first parent or
precursor ions and the second parent or precursor ions which are
being compared may be determined to be multiply charged. This may
rule out a number of fragment, product, daughter or adduct ions
which quite often tend to be singly charged. The first parent or
precursor ions and the second parent or precursor ions may
according to a more preferred embodiment be determined to have the
same charge state. According to another embodiment, the parent or
precursor ions being compared in the two different samples may be
determined to give rise to fragment, product, daughter or adduct
ions which have the same charge state.
[0046] The first sample and/or the second sample may comprise a
plurality of different biopolymers, proteins, peptides,
polypeptides, oligionucleotides, oligionucleosides, amino acids,
carbohydrates, sugars, lipids, fatty acids, vitamins, hormones,
portions or fragments of DNA, portions or fragments of cDNA,
portions or fragments of RNA, portions or fragments of mRNA,
portions or fragments of tRNA, polyclonal antibodies, monoclonal
antibodies, ribonucleases, enzymes, metabolites, polysaccharides,
phosphorylated peptides, phosphorylated proteins, glycopeptides,
glycoproteins or steroids. The first sample and/or the second
sample may also comprise at least 2, 5, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500, or 5000 molecules having
different identities.
[0047] The first sample may be taken from a diseased organism and
the second sample may be taken from a non-diseased organism.
Alternatively, the first sample may be taken from a treated
organism and the second sample may be taken from a non-treated
organism. According to another embodiment the first sample may be
taken from a mutant organism and the second sample may be taken
from a wild type organism.
[0048] Molecules from the first and/or second samples are
preferably separated from a mixture of other molecules prior to
being ionised by High Performance Liquid Chromatography ("HPLC"),
anion exchange, anion exchange chromatography, cation exchange,
cation exchange chromatography, ion pair reversed-phase
chromatography, chromatography, single dimensional electrophoresis,
multi-dimensional electrophoresis, size exclusion, affinity,
reverse phase chromatography, Capillary Electrophoresis
Chromatography ("CEC"), electrophoresis, ion mobility separation,
Field Asymmetric Ion Mobility Separation ("FAIMS") or capillary
electrophoresis.
[0049] According to a particularly preferred embodiment the first
and second sample ions may comprise peptide ions. The peptide ions
preferably comprise the digest products of one or more proteins. An
attempt may be made to identify a protein which correlates with
parent peptide ions of interest. Preferably, a determination is
made as to which peptide products are predicted to be formed when a
protein is digested and it is then determined whether any predicted
peptide product(s) correlate with parent or precursor ions of
interest. A determination may also be made as to whether the parent
or precursor ions of interest correlate with one or more
proteins.
[0050] The first and second samples may be taken from the same
organism or from different organisms.
[0051] A check may be made to confirm that the first and second
parent or precursor ions being compared really are parent or
precursor ions rather than fragment, product, daughter or adduct
ions. A high fragmentation mass spectrum relating to data obtained
in the high fragmentation or reaction mode may be compared with a
low fragmentation mass spectrum relating to data obtained in the
low fragmentation or reaction mode wherein the mass spectra were
obtained at substantially the same time. A determination may be
made that the first and/or the second parent or precursor ions are
not fragment, product, daughter or adduct ions if the first and/or
the second parent or precursor ions have a greater intensity in the
low fragmentation mass spectrum relative to the high fragmentation
mass spectrum. Similarly, fragment, product, daughter or adduct
ions may be recognised by noting ions having a greater intensity in
the high fragmentation mass spectrum relative to the low
fragmentation mass spectrum.
[0052] Parent or precursor ions from the first sample and parent or
precursor ions from the second sample are preferably passed to the
same collision, fragmentation or reaction device. However,
according to a less preferred embodiment, parent or precursor ions
from the first sample and parent or precursor ions from the second
sample may be passed to different collision, fragmentation or
reaction devices.
[0053] According to another aspect of the present invention there
is provided a mass spectrometer comprising:
[0054] a collision, fragmentation or reaction device repeatedly
switched, altered or varied in use between a first mode wherein at
least some parent or precursor ions are fragmented into one or more
fragment, product, daughter or adduct ions and a second mode
wherein substantially fewer parent or precursor ions are fragmented
or reacted;
[0055] a mass analyser; and
[0056] a control system which in use:
[0057] (i) determines the intensity of first parent or precursor
ions from a first sample which have a first mass to charge
ratio;
[0058] (ii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio; and
[0059] (iii) compares the intensity of the first parent or
precursor ions with the intensity of the second parent or precursor
ions;
[0060] wherein if the intensity of the first parent or precursor
ions differs from the intensity of the second parent or precursor
ions by more than a predetermined amount then either the first
parent or precursor ions and/or the second parent or precursor ions
are considered to be parent or precursor ions of interest; and
[0061] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0062] According to another aspect of the invention there is
provided a mass spectrometer comprising:
[0063] a collision, fragmentation or reaction device repeatedly
switched, altered or varied in use between a first mode wherein at
least some parent or precursor ions are fragmented or reacted into
one or more fragment, product, daughter or adduct ions and a second
mode wherein substantially fewer parent or precursor ions are
fragmented or reacted;
[0064] a mass analyser; and
[0065] a control system which in use:
[0066] (i) determines the intensity of first parent or precursor
ions from a first sample which have a first mass to charge
ratio;
[0067] (ii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio;
[0068] (iii) determines a first ratio of the intensity of the first
parent or precursor ions to the intensity of other parent or
precursor ions in the first sample;
[0069] (iv) determines a second ratio of the intensity of the
second parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
[0070] (v) compares the first ratio with the second ratio;
[0071] wherein if the first ratio differs from the second ratio by
more than a predetermined amount then either the first parent or
precursor ions and/or the second parent or precursor ions are
considered to be parent or precursor ions of interest; and
[0072] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0073] The mass spectrometer preferably further comprises an ion
source. The ion source is preferably selected from the group
consisting of: (i) an Electrospray ionisation ("ESI") ion source;
(ii) an Atmospheric Pressure Photo Ionisation ("APPI") ion source;
(iii) an Atmospheric Pressure Chemical Ionisation ("APCI") ion
source; (iv) a Matrix Assisted Laser Desorption Ionisation
("MALDI") ion source; (v) a Laser Desorption Ionisation ("LDI") ion
source; (vi) an Atmospheric Pressure Ionisation ("API") ion source;
(vii) a Desorption Ionisation on Silicon ("DIOS") ion source;
(viii) an Electron Impact ("EI") ion source; (ix) a Chemical
Ionisation ("CI") ion source; (x) a Field Ionisation ("FI") ion
source; (xi) a Field Desorption ("FD") ion source; (xii) an
Inductively Coupled Plasma ("ICP") ion source; (xiii) a Fast Atom
Bombardment ("FAB") ion source; (xiv) a Liquid Secondary Ion Mass
Spectrometry ("LSIMS") ion source; (xv) a Desorption Electrospray
Ionisation ("DESI") ion source; (xvi) a Nickel-63 radioactive ion
source; (xvii) an Atmospheric Pressure Matrix Assisted Laser
Desorption Ionisation ion source; and (xviii) a Thermospray ion
source.
[0074] The ion source may comprise a pulsed or a continuous ion
source.
[0075] According to an embodiment the mass spectrometer may
comprise an Electrospray, Atmospheric Pressure Chemical Ionisation
("APCI"), Atmospheric Pressure Photo Ionisation ("APPI"), Matrix
Assisted Laser Desorption Ionisation ("MALDI"), Laser Desorption
Ionisation ("LDI"), Inductively Coupled Plasma ("ICP"), Fast Atom
Bombardment ("FAB") or Liquid Secondary Ions Mass Spectrometry
("LSIMS") ion source. Such ion sources may be provided with an
eluent over a period of time, the eluent having been separated from
a mixture by means of liquid chromatography or capillary
electrophoresis.
[0076] Alternatively, the mass spectrometer may comprise an
Electron Impact ("EI"), Chemical Ionisation ("CI") or Field
Ionisation ("FI") ion source. Such ion sources may be provided with
an eluent over a period of time, the eluent having been separated
from a mixture by means of gas chromatography.
[0077] The mass analyser preferably comprises a quadrupole mass
filter, a Time of Flight ("TOF") mass analyser (an orthogonal
acceleration Time of Flight mass analyser is particularly
preferred), a 2D (linear) or 3D (doughnut shaped electrode with two
endcap electrodes) ion trap, a magnetic sector analyser or a
Fourier Transform Ion Cyclotron Resonance ("FTICR") mass
analyser.
[0078] The collision, fragmentation or reaction device may comprise
a quadrupole rod set, an hexapole rod set, an octopole or higher
order rod set or an ion tunnel comprising a plurality of electrodes
having apertures through which ions are transmitted. The apertures
are preferably substantially the same size. The collision,
fragmentation or reaction device may, more generally, comprise a
plurality of electrodes connected to an AC or RF voltage supply for
radially confining ions within the collision, fragmentation or
reaction device. An axial DC voltage gradient may or may not be
applied along at least a portion of the length of the ion tunnel
collision, fragmentation or reaction device. The collision,
fragmentation or reaction device may be housed in a housing or
otherwise arranged so that a substantially gas-tight enclosure is
formed around the collision, fragmentation or reaction device apart
from an aperture to admit ions and an aperture for ions to exit
from and optionally a port for introducing gas. A gas such as
helium, argon, nitrogen, air or methane may be introduced into the
collision, fragmentation or reaction device.
[0079] Other arrangements are also contemplated wherein the
collision, fragmentation or reaction device is not repeatedly
switched, altered or varied between a high fragmentation or
reaction mode and a low fragmentation or reaction mode. For
example, the collision, fragmentation or reaction device may be
left permanently ON and arranged to fragment or react ions received
within the collision, fragmentation or reaction device. An
electrode or other device may be provided upstream of the
collision, fragmentation or reaction device. A high fragmentation
or reaction mode of operation would occur when the electrode or
other device allowed ions to pass to the collision, fragmentation
or reaction device. A low fragmentation or reaction mode of
operation would occur when the electrode or other device caused
ions to by-pass the collision, fragmentation or reaction device and
hence not be fragmented or reacted therein.
[0080] Other embodiments are also contemplated which would be
useful where particular parent or precursor ions could not be
easily observed since they co-eluted with other commonly observed
peptide ions. In such circumstances the expression level of
fragment, product, daughter or adduct ions is compared between two
samples.
[0081] According to another aspect of the invention there is
provided a method of mass spectrometry comprising:
[0082] passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device;
[0083] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the first sample
are fragmented or reacted into one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0084] passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device;
[0085] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the second sample
are fragmented or reacted into one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0086] automatically determining the intensity of first fragment,
product, daughter or adduct ions derived from first parent or
precursor ions from the first sample, the first fragment, product,
daughter or adduct ions having a first mass to charge ratio;
[0087] automatically determining the intensity of second fragment,
product, daughter or adduct ions derived from second parent or
precursor ions from the second sample, the second fragment,
product, daughter or adduct ions having the same first mass to
charge ratio; and
[0088] comparing the intensity of the first fragment, product,
daughter or adduct ions with the intensity of the second fragment,
product, daughter or adduct ions;
[0089] wherein if the intensity of the first fragment, product,
daughter or adduct ions differs from the intensity of the second
fragment, product, daughter or adduct ions by more than a
predetermined amount then either the first parent or precursor ions
and/or the second parent or precursor ions are considered to be
parent or precursor ions of interest; and
[0090] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0091] In a similar manner, according to another aspect of the
invention there is provided a method of mass spectrometry
comprising:
[0092] passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device;
[0093] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the first sample
are fragmented or reacted into one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0094] passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device;
[0095] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the second sample
are fragmented or reacted into one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0096] automatically determining the intensity of first fragment,
product, daughter or adduct ions derived from first parent or
precursor ions from the first sample, the first fragment, product,
daughter or adduct ions having a first mass to charge ratio;
[0097] automatically determining the intensity of second fragment,
product, daughter or adduct ions derived from second parent or
precursor ions from the second sample, the second fragment,
product, daughter or adduct ions having the same first mass to
charge ratio;
[0098] determining a first ratio of the intensity of the first
fragment, product, daughter or adduct ions to the intensity of
other parent or precursor ions in the first sample or with the
intensity of other fragment, product, daughter or adduct ions
derived from other parent or precursor ions in the first
sample;
[0099] determining a second ratio of the intensity of the second
fragment, product, daughter or adduct ions to the intensity of
other parent or precursor ions in the second sample or with the
intensity of other fragment, product, daughter or adduct ions
derived from other parent or precursor ions in the second
sample;
[0100] comparing the first ratio with the second ratio;
[0101] wherein if the first ratio differs from the second ratio by
more than a predetermined amount then either the first parent or
precursor ions and/or the second parent or precursor ions are
considered to be parent or precursor ions of interest; and
[0102] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0103] According to another aspect of the invention there is
provided a mass spectrometer comprising:
[0104] a collision, fragmentation or reaction device repeatedly
switched, altered or varied in use between a first mode wherein at
least some parent or precursor ions are fragmented or reacted into
one or more fragment, product, daughter or adduct ions and a second
mode wherein substantially fewer parent or precursor ions are
fragmented;
[0105] a mass analyser; and
[0106] a control system which in use:
[0107] (i) determines the intensity of first fragment, product,
daughter or adduct ions derived from first parent or precursor ions
from a first sample, the first fragment, product, daughter or
adduct ions having a first mass to charge ratio;
[0108] (ii) determines the intensity of second fragment, product,
daughter or adduct ions derived from second parent or precursor
ions from a second sample, the second fragment, product, daughter
or adduct ions having the same first mass to charge ratio; and
[0109] (iii) compares the intensity of the first fragment, product,
daughter or adduct ions with the intensity of the second fragment,
product, daughter or adduct ions;
[0110] wherein if the intensity of the first fragment, product,
daughter or adduct ions differs from the intensity of the second
fragment, product, daughter or adduct ions by more than a
predetermined amount then either the first parent or precursor ions
and/or the second parent or precursor ions are considered to be
parent or precursor ions of interest; and
[0111] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0112] According to another aspect of the invention there is
provided a mass spectrometer comprising:
[0113] a collision, fragmentation or reaction device repeatedly
switched, altered or varied in use between a first mode wherein at
least some parent or precursor ions are fragmented or reacted into
one or more fragment, product, daughter or adduct ions and a second
mode wherein substantially fewer parent or precursor ions are
fragmented or reacted;
[0114] a mass analyser; and
[0115] a control system which in use:
[0116] (i) determines the intensity of first fragment, product,
daughter or adduct ions derived from first parent or precursor ions
from a first sample, the first fragment, product, daughter or
adduct ions having a first mass to charge ratio;
[0117] (ii) determines the intensity of second fragment, product,
daughter or adduct ions derived from second parent or precursor
ions from a second sample, the second fragment, product, daughter
or adduct ions having the same first mass to charge ratio;
[0118] (iii) determines a first ratio of the intensity of the first
fragment, product, daughter or adduct ions to the intensity of
other parent or precursor ions in the first sample or with the
intensity of other fragment, product, daughter or adduct ions
derived from other parent or precursor ions in the first
sample;
[0119] (iv) determines a second ratio of the intensity of the
second fragment, product, daughter or adduct ions to the intensity
of other parent or precursor ions in the second sample or with the
intensity of other fragment, product, daughter or adduct ions
derived from other parent or precursor ions in the second sample;
and
[0120] (v) compares the first ratio with the second ratio;
[0121] wherein if the first ratio differs from the second ratio by
more than a predetermined amount then either the first parent or
precursor ions and/or the second parent or precursor ions are
considered to be parent or precursor ions of interest; and
[0122] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0123] It will be apparent that the above described embodiments
which relate to comparing the expression level of fragment,
product, daughter or adduct ions rather than parent or precursor
ions either directly or indirectly may employ the method and
apparatus relating to the preferred embodiment. Therefore, the same
preferred features which are recited with respect to the preferred
embodiment may also be used with the embodiments which relate to
comparing the expression level of fragment, product, daughter or
adduct ions.
[0124] An arrangement is contemplated wherein instead of comparing
the expression levels of parent or precursor ions in two different
samples and seeing whether the expression levels are significantly
different so as to warrant further investigation, an initial
recognition may instead be made that parent or precursor ions of
interest are present in a sample.
[0125] According to this arrangement there is provided a method of
mass spectrometry comprising:
[0126] passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device;
[0127] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the first sample
are fragmented or reacted into one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0128] passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device;
[0129] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the second sample
are fragmented or reacted into one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0130] recognising first parent or precursor ions of interest from
the first sample;
[0131] automatically determining the intensity of the first parent
or precursor ions of interest, the first parent or precursor ions
of interest having a first mass to charge ratio;
[0132] automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first
mass to charge ratio; and
[0133] comparing the intensity of the first parent or precursor
ions of interest with the intensity of the second parent or
precursor ions; and
[0134] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0135] According to another aspect of the invention, there is
provided a method of mass spectrometry comprising:
[0136] passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device;
[0137] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the first sample
are fragmented or reacted into one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0138] passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device;
[0139] repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the second sample
are fragmented or reacted into one or more fragment, product,
daughter or adduct ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented or reacted;
[0140] recognising first parent or precursor ions of interest from
the first sample;
[0141] automatically determining the intensity of the first parent
or precursor ions of interest, the first parent or precursor ions
of interest having a first mass to charge ratio;
[0142] automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first
mass to charge ratio;
[0143] determining a first ratio of the intensity of the first
parent or precursor ions of interest to the intensity of other
parent or precursor ions in the first sample;
[0144] determining a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
[0145] comparing the first ratio with the second ratio; and
[0146] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0147] It is apparent that the same preferred features which are
described above in relation to the preferred embodiment may also be
provided in relation to above arrangement and hence will not be
repeated.
[0148] According to a preferred embodiment, the step of recognising
first parent or precursor ions of interest comprises recognising
first fragment, product, daughter or adduct ions of interest.
[0149] The first fragment, product, daughter or adduct ions of
interest may be optionally identified by, for example, determining
their mass to charge ratio preferably to less than or equal to 20
ppm, 15 ppm, 10 ppm or 5 ppm.
[0150] Having recognised and optionally identified fragment,
product, daughter or adduct ions of interest, it is then necessary
to determine which parent or precursor ion gave rise to that
fragment, product, daughter or adduct ion.
[0151] The step of recognising first parent or precursor ions of
interest may comprise determining whether parent or precursor ions
are observed in a mass spectrum obtained when the collision,
fragmentation or reaction device is in the low fragmentation or
reaction mode for a certain time period and first fragment,
product, daughter or adduct ions of interest are observed in a mass
spectrum obtained either immediately before the certain time
period, when the collision, fragmentation or reaction device is in
the high fragmentation or reaction mode, or immediately after the
certain time period, when the collision, fragmentation or reaction
device is in the high fragmentation or reaction mode.
[0152] The step of recognising first parent or precursor ions of
interest may comprise comparing the elution times of parent or
precursor ions with the pseudo-elution time of first fragment,
product, daughter or adduct ions of interest. The step of
recognising first parent or precursor ions of interest may also
comprise comparing the elution profiles of parent or precursor ions
with the pseudo-elution profile of first fragment, product,
daughter or adduct ions of interest.
[0153] According to another less preferred embodiment, parent or
precursor ions of interest may be recognised immediately by virtue
of their mass to charge ratio without it being necessary to
recognise and identify fragment, product, daughter or adduct ions
of interest. According to this embodiment the step of recognising
first parent or precursor ions of interest preferably comprises
determining the mass to charge ratio of the parent or precursor
ions preferably to less than or equal to 20 ppm, 15 ppm, 10 ppm or
5 ppm. The determined mass to charge ratio of the parent or
precursor ions may then be compared with a database of ions and
their corresponding mass to charge ratios.
[0154] According to another embodiment, the step of recognising
first parent or precursor ions of interest comprises determining
whether parent or precursor ions give rise to fragment, product,
daughter or adduct ions as a result of the loss of a predetermined
ion or a predetermined neutral particle.
[0155] Parent or precursor ions of interest may be identified in a
similar manner to the first main embodiment.
[0156] The other preferred features of the preferred embodiment
apply equally to the other arrangement.
[0157] According to another arrangement there is provided a mass
spectrometer comprising:
[0158] a collision, fragmentation or reaction device repeatedly
switched, altered or varied in use between a first mode wherein at
least some parent or precursor ions are fragmented or reacted into
one or more fragment, product, daughter or adduct ions and a second
mode wherein substantially fewer parent or precursor ions are
fragmented or reacted;
[0159] a mass analyser; and
[0160] a control system which in use:
[0161] (i) recognises first parent or precursor ions of interest
from a first sample, the first parent or precursor ions of interest
having a first mass to charge ratio;
[0162] (ii) determines the intensity of the first parent or
precursor ions of interest;
[0163] (iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio; and
[0164] (iv) compares the intensity of the first parent or precursor
ions of interest with the intensity of the second parent or
precursor ions; and
[0165] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0166] According to another arrangement there is provided a mass
spectrometer comprising:
[0167] a collision, fragmentation or reaction device repeatedly
switched, altered or varied in use between a first mode wherein at
least some parent or precursor ions are fragmented or reacted into
one or more fragment, product, daughter or adduct ions and a second
mode wherein substantially fewer parent or precursor ions are
fragmented or reacted;
[0168] a mass analyser; and
[0169] a control system which in use:
[0170] (i) recognises first parent or precursor ions of interest
from a first sample, the first parent or precursor ions of interest
having a first mass to charge ratio;
[0171] (ii) determines the intensity of the first parent or
precursor ions of interest;
[0172] (iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio;
[0173] (iv) determines a first ratio of the intensity of the first
parent or precursor ions of interest to the intensity of other
parent or precursor ions in the first sample;
[0174] (v) determines a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
[0175] (vi) compares the first ratio with the second ratio;
[0176] wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) a Surface Induced
Dissociation ("SID") collision, fragmentation or reaction device;
(ii) an Electron Transfer Dissociation collision, fragmentation or
reaction device; (iii) an Electron Capture Dissociation collision,
fragmentation or reaction device; (iv) an Electron Collision or
Impact Dissociation collision, fragmentation or reaction device;
(v) a Photo Induced Dissociation ("PID") collision, fragmentation
or reaction device; (vi) a Laser Induced Dissociation collision,
fragmentation or reaction device; (vii) an infrared radiation
induced dissociation device; (viii) an ultraviolet radiation
induced dissociation device; (ix) a nozzle-skimmer interface
collision, fragmentation or reaction device; (x) an in-source
collision, fragmentation or reaction device; (xi) an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device; (xii) a thermal or temperature source collision,
fragmentation or reaction device; (xiii) an electric field induced
collision, fragmentation or reaction device; (xiv) a magnetic field
induced collision, fragmentation or reaction device; (xv) an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device; (xvi) an ion-ion reaction collision, fragmentation
or reaction device; (xvii) an ion-molecule reaction collision,
fragmentation or reaction device; (xviii) an ion-atom reaction
collision, fragmentation or reaction device; (xix) an
ion-metastable ion reaction collision, fragmentation or reaction
device; (xx) an ion-metastable molecule reaction collision,
fragmentation or reaction device; (xxi) an ion-metastable atom
reaction collision, fragmentation or reaction device; (xxii) an
ion-ion reaction device for reacting ions to form adduct or product
ions; (xxiii) an ion-molecule reaction device for reacting ions to
form adduct or product ions; (xxiv) an ion-atom reaction device for
reacting ions to form adduct or product ions; (xxv) an
ion-metastable ion reaction device for reacting ions to form adduct
or product ions; (xxvi) an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions; and (xxvii) an
ion-metastable atom reaction device for reacting ions to form
adduct or product ions.
[0177] It will be apparent that the above described embodiments
which relate to recognising parent or precursor ions of interest
and comparing the expression level of parent or precursor ions of
interest in one sample with corresponding parent or precursor ions
in another sample may employ the method and apparatus relating to
the preferred embodiment. Therefore, the same preferred features
which are recited with respect to the preferred embodiment may also
be used with the embodiments which relate to recognising parent or
precursor ions of interest and then comparing the expression level
of the parent or precursor ions of interest in one sample with
corresponding parent or precursor ions in another sample.
[0178] If parent or precursor ions having a particular mass to
charge ratio are expressed differently in two different samples,
then according to the preferred embodiment further investigation of
the parent or precursor ions of interest then occurs. This further
investigation may comprise seeking to identify the parent or
precursor ions of interest which are expressed differently in the
two different samples. In order to verify that the parent or
precursor ions whose expression levels are being compared in the
two different samples really are the same ions, a number of checks
may be made.
[0179] Measurements of changes in the abundance of proteins in
complex protein mixtures can be extremely informative. For example,
changes to the abundance of proteins in cells, often referred to as
the protein expression level, could be due to different cellular
stresses, the effect of stimuli, the effect of disease or the
effect of drugs. Such proteins may provide relevant targets for
study, screening or intervention. The identification of such
proteins will normally be of interest. Such proteins may be
identified by the method of the preferred embodiment.
[0180] Therefore, according to the preferred embodiment a new
criterion for the discovery of parent or precursor ions of interest
is based on the quantification of proteins in two different
samples. This requires the determination of the relative abundances
of their peptide products in two or more samples. However, the
determination of relative abundance requires that the same peptide
ions must be compared in the two (or more) different samples and
ensuring that this happens is a non-trivial problem. Hence, it is
necessary to be able to recognise and preferably identify the
peptide ion to the extent that it can at least be uniquely
recognised within the sample. Such peptide ions may be adequately
recognised by measurement of the mass of the parent or precursor
ion and by measurement of the mass to charge ratio of one or more
fragment, product, daughter or adduct ions derived from that parent
or precursor ion. The specificity with which the peptides may be
recognised may be increased by the determination of the accurate
mass of the parent or precursor ion and/or the accurate mass of one
or more fragment, product, daughter or adduct ions.
[0181] The same method of recognising parent or precursor ions in
one sample is also preferably used to recognise the same parent or
precursor ions in another sample and this enables the relative
abundances of the parent or precursor ions in the two different
samples to be measured.
[0182] Measurement of relative abundances allows discovery of
proteins with a significant change or difference in expression
level of that protein. The same data allows identification of that
protein by the method already described in which several or all
fragment, product, daughter or adduct ions associated with each
such peptide product ion is discovered by closeness of fit of their
respective elution times. Again, the accurate measurement of the
masses of the parent or precursor ion and associated fragment,
product, daughter or adduct ions substantially improves the
specificity and confidence with which the protein may be
identified.
[0183] The specificity with which the peptides may be recognised
may also be increased by comparison of retention times. For
example, the HPLC or CE retention or elution times will be measured
as part of the procedure for associating fragment, product,
daughter or adduct ions with parent or precursor ions, and these
elution times may also be compared for the two or more samples. The
elution times may be used to reject measurements where they do not
fall within a pre-defined time difference of each other.
Alternatively, retention times may be used to confirm recognition
of the same peptide when they do fall within a predefined window of
each other. Commonly there may be some redundancy if the parent or
precursor ion accurate mass, one or more fragment, product,
daughter or adduct ion accurate masses, and the retention times are
all measured and compared. In many instances just two of these
measurements will be adequate to recognise the same peptide parent
or precursor ion in the two or more samples. For example,
measurement of just the accurate parent or precursor ion mass to
charge ratio and a fragment, product, daughter or adduct ion mass
to charge ratio, or the accurate parent or precursor ion mass to
charge ratio and the retention time, may well be adequate.
Nevertheless, the additional measurements may be used to confirm
the recognition of the same parent peptide ion.
[0184] The relative expression levels of the matched parent peptide
ions may be quantified by measuring the peak areas relative to an
internal standard.
[0185] The preferred embodiment does not require any interruption
to the acquisition of data and hence is particularly suitable for
quantitative applications. According to an embodiment one or more
endogenous peptides common to both mixtures which are not changed
by the experimental state of the samples may used as an internal
standard or standards for the relative peak area measurements.
According to another embodiment an internal standard may be added
to each sample where no such internal standard is present or can be
relied upon. The internal standard, whether naturally present or
added, may also serve as a chromatographic retention time standard
as well as a mass accuracy standard.
[0186] Ideally more than one peptide parent or precursor ion may be
measured for each protein to be quantified. For each peptide the
same means of recognition is preferably used when comparing
intensities in each of the different samples. The measurements of
different peptides serves to validate the relative abundance
measurements. Furthermore, the measurements from several peptides
provides a means of determining the average relative abundance, and
of determining the relative significance of the measurements.
[0187] According to one embodiment all parent or precursor ions may
be identified and their relative abundances determined by
comparison of their intensities to those of the same identity in
one or more other samples.
[0188] In another embodiment the relative abundance of all parent
or precursor ions of interest, discovered on the basis of their
relationship to a predetermined fragment, product, daughter or
adduct ion, may be determined by comparison of their intensities to
those of the same identity in one or more other samples.
[0189] In another embodiment the relative abundance of all parent
or precursor ions of interest, discovered on the basis of their
giving rise to a predetermined mass loss, may be determined by
comparison of their intensities to those of the same identity in
one or more other samples.
[0190] In another embodiment it may be merely required to quantify
a protein already identified. The protein may be in a complex
mixture, and the same means for separation and recognition may be
used as that already described. Here it is only necessary to
recognise the relevant peptide product or products and measure
their intensities in one or more samples. The basis for recognition
may be that of the peptide parent or precursor ion mass or accurate
mass, and that of one or more fragment, product, daughter or adduct
ion masses, or accurate masses. Their retention times may also be
compared thereby providing a means of confirming the recognition of
the same peptide or of rejecting unmatched peptides.
[0191] The preferred embodiment is applicable to the study of
proteomics. However, the same methods of identification and
quantification may be used in other areas of analysis such as the
study of metabolomics.
[0192] The method is appropriate for the analysis of mixtures where
different components of the mixture are first separated or
partially separated by a means such as chromatography that causes
components to elute sequentially.
[0193] The source of ions may preferably yield mainly molecular
ions or pseudo-molecular ions and relatively few (if any) fragment,
product, daughter or adduct ions. Examples of such sources include
atmospheric pressure ionisation sources (e.g. Electrospray and
APCI) and Matrix Assisted Laser Desorption Ionisation (MALDI).
[0194] The collision, fragmentation or reaction device may comprise
a chamber containing gas at a sufficient density to ensure that all
the ions collide with gas molecules at least once during their
transit through the chamber. If the collision energy is set low by
using low voltages the collisions do not induce fragmentation. If
the collision energy is increased sufficiently then collisions will
start to induce fragmentation. The fragmentation ions are also
known as fragment, product, daughter or adduct ions. The collision,
fragmentation or reaction device is preferably operated in at least
two distinct operating modes - a first mode, wherein many or most
of the sample or parent or precursor ions are fragmented or reacted
to produce fragment, product, daughter or adduct ions and a second
mode, wherein none or very few of the sample or product ions are
fragmented or reacted.
[0195] If the two main operating modes are suitably set, then
parent or precursor ions can be recognised by virtue of the fact
that they will be relatively more intense in the mass spectrum
without substantial fragmentation or reaction. Similarly, fragment,
product, daughter or adduct ions can be recognised by virtue of the
fact that they will be relatively more intense in the mass spectrum
with substantial fragmentation or reaction.
[0196] The mass analyser may comprise a quadrupole, Time of Flight,
ion trap, magnetic sector or FT-ICR mass analyser. According to a
preferred embodiment the mass analyser should be capable of
determining the exact or accurate mass to charge value for ions.
This is to maximise selectivity for detection of characteristic
fragment, product, daughter or adduct ions or mass losses, and to
maximise specificity for identification of proteins.
[0197] The mass analyser preferably samples or records the whole
spectrum simultaneously. This ensures that the elution times
observed for all the masses are not modified or distorted by the
mass analyser, and in turn would allow accurate matching of the
elution times of different masses, such as parent and fragment,
product, daughter or adduct ions. It also helps to ensure that the
quantitative measurements are not compromised by the need to
measure abundances of transient signals.
[0198] A mass filter, preferably a quadrupole mass filter, may be
provided upstream of the collision, fragmentation or reaction
device. The mass filter may have a highpass filter characteristic
and, for example, be arranged to transmit ions having a mass to
charge ratio greater than or equal to 100, 150, 200, 250, 300, 350,
400, 450 or 500. Alternatively, the mass filter may have a lowpass
or bandpass filter characteristic.
[0199] An ion guide may be provided upstream of the collision,
fragmentation or reaction device. The ion guide may comprise either
a hexapole, quadrupole, octopole or higher order multipole rod set.
In another embodiment the ion guide may comprise an ion tunnel ion
guide comprising a plurality of electrodes having apertures through
which ions are transmitted in use. Preferably, at least 90% of the
electrodes have apertures which are substantially the same size.
Alternatively, the ion guide may comprise a plurality of ring
electrodes having substantially tapering internal diameters ("ion
funnel").
[0200] Parent or precursor ions that belong to a particular class
of parent or precursor ions, and which are recognisable by a
characteristic fragment, product, daughter or adduct ion or
characteristic neutral loss are traditionally discovered by the
methods of parent or precursor ion scanning or constant neutral
loss scanning. Previous methods for recording parent or precursor
ion scans or constant neutral loss scans involve scanning one or
both quadrupoles in a triple quadrupole mass spectrometer, or
scanning the quadrupole in a tandem quadrupole orthogonal TOF mass
spectrometer, or scanning at least one element in other types of
tandem mass spectrometers. As a consequence, these methods suffer
from the low duty cycle associated with scanning instruments. As a
further consequence, information may be discarded and lost whilst
the mass spectrometer is occupied recording a parent or precursor
ion scan or a constant neutral loss scan. As a further consequence
these methods are not appropriate for use where the mass
spectrometer is required to analyse substances eluting directly
from gas or liquid chromatography equipment.
[0201] According to the preferred embodiment, a tandem quadrupole
orthogonal TOF mass spectrometer in used in a way in which parent
or precursor ions of interest are discovered using a method in
which sequential low and high collision energy mass spectra are
recorded. The switching, altering or varying back and forth is
preferably not interrupted. Instead a complete set of data is
acquired, and this is then processed afterwards. Fragment, product,
daughter or adduct ions may be associated with parent or precursor
ions by closeness of fit of their respective elution times. In this
way parent or precursor ions of interest may be confirmed or
otherwise without interrupting the acquisition of data, and
information need not be lost.
[0202] According to one embodiment, possible parent or precursor
ions of interest may be selected on the basis of their relationship
to a predetermined fragment, product, daughter or adduct ion. The
predetermined fragment, product, daughter or adduct ion may
comprise, for example, immonium ions from peptides, functional
groups including phosphate group PO.sub.3.sup.- ions from
phosphorylated peptides or mass tags which are intended to cleave
from a specific molecule or class of molecule and to be
subsequently identified thus reporting the presence of the specific
molecule or class of molecule. A parent or precursor ion may be
short listed as a possible parent or precursor ion of interest by
generating a mass chromatogram for the predetermined fragment,
product, daughter or adduct ion using high fragmentation or
reaction mass spectra. The centre of each peak in the mass
chromatogram is then determined together with the corresponding
predetermined fragment, product, daughter or adduct ion elution
time(s). Then for each peak in the predetermined fragment, product,
daughter or adduct ion mass chromatogram both the low fragmentation
or reaction mass spectrum obtained immediately before the
predetermined fragment, product, daughter or adduct ion elution
time and the low fragmentation or reaction mass spectrum obtained
immediately after the predetermined fragment, product, daughter or
adduct ion elution time are interrogated for the presence of
previously recognised parent or precursor ions. A mass chromatogram
for any previously recognised parent or precursor ion found to be
present in both the low fragmentation or reaction mass spectrum
obtained immediately before the predetermined fragment, product,
daughter or adduct ion elution time and the low fragmentation or
reaction mass spectrum obtained immediately after the predetermined
fragment, product, daughter or adduct ion elution time is then
generated and the centre of each peak in each mass chromatogram is
determined together with the corresponding possible parent or
precursor ion of interest elution time(s). The possible parent or
precursor ions of interest may then be ranked according to the
closeness of fit of their elution time with the predetermined
fragment, product, daughter or adduct ion elution time, and a list
of final possible parent or precursor ions of interest may be
formed by rejecting possible parent or precursor ions of interest
if their elution time precedes or exceeds the predetermined
fragment, product, daughter or adduct ion elution time by more than
a predetermined amount.
[0203] According to an alternative embodiment, a parent or
precursor ion may be shortlisted as a possible parent or precursor
ion of interest on the basis of it giving rise to a predetermined
mass loss. For each low fragmentation or reaction mass spectrum, a
list of target fragment, product, daughter or adduct ion mass to
charge values that would result from the loss of a predetermined
ion or neutral particle from each previously recognised parent or
precursor ion present in the low fragmentation or reaction mass
spectrum is generated. Then both the high fragmentation or reaction
mass spectrum obtained immediately before the low fragmentation or
reaction mass spectrum and the high fragmentation or reaction mass
spectrum obtained immediately after the low fragmentation or
reaction mass spectrum are interrogated for the presence of
fragment, product, daughter or adduct ions having a mass to charge
value corresponding with a target fragment, product, daughter or
adduct ion mass to charge value. A list of possible parent or
precursor ions of interest (optionally including their
corresponding fragment, product, daughter or adduct ions) is then
formed by including in the list a parent or precursor ion if a
fragment, product, daughter or adduct ion having a mass to charge
value corresponding with a target fragment, product, daughter or
adduct ion mass to charge value is found to be present in both the
high fragmentation or reaction mass spectrum immediately before the
low fragmentation or reaction mass spectrum and the high
fragmentation or reaction mass spectrum immediately after the low
fragmentation or reaction mass spectrum. A mass loss chromatogram
may then be generated based upon possible candidate parent or
precursor ions and their corresponding fragment, product, daughter
or adduct ions. The centre of each peak in the mass loss
chromatogram is determined together with the corresponding mass
loss elution time(s). Then for each possible candidate parent or
precursor ion a mass chromatogram is generated using the low
fragmentation or reaction mass spectra. A corresponding fragment,
product, daughter or adduct ion mass chromatogram is also generated
for the corresponding fragment, product, daughter or adduct ion.
The centre of each peak in the possible candidate parent or
precursor ion mass chromatogram and the corresponding fragment,
product, daughter or adduct ion mass chromatogram are then
determined together with the corresponding possible candidate
parent or precursor ion elution time(s) and corresponding fragment,
product, daughter or adduct ion elution time(s). A list of final
candidate parent or precursor ions may then be formed by rejecting
possible candidate parent or precursor ions if the elution time of
a possible candidate parent or precursor ion precedes or exceeds
the corresponding fragment, product, daughter or adduct ion elution
time by more than a predetermined amount.
[0204] Once a list of parent or precursor ions of interest has been
formed (which preferably comprises only some of the originally
recognised parent or precursor ions and possible parent or
precursor ions of interest) then each parent or precursor ion of
interest can then be identified.
[0205] Identification of parent or precursor ions may be achieved
by making use of a combination of information. This may include the
accurately determined mass or mass to charge ratio of the parent or
precursor ion. It may also include the masses or mass to charge
ratios of the fragment, product, daughter or adduct ions. In some
instances the accurately determined masses or mass to charge ratios
of the fragment, product, daughter or adduct ions may be preferred.
It is known that a protein may be identified from the masses or
mass to charge ratios, preferably the exact masses, of the peptide
products from proteins that have been enzymatically digested. These
may be compared to those expected from a library of known proteins.
It is also known that when the results of this comparison suggest
more than one possible protein then the ambiguity can be resolved
by analysis of the fragments of one or more of the peptides. The
preferred embodiment allows a mixture of proteins, which have been
enzymatically digested, to be identified in a single analysis. The
masses or mass to charge ratios, or exact masses or mass to charge
ratios, of all the peptides and their associated fragment, product,
daughter or adduct ions may be searched against a library of known
proteins. Alternatively, the peptide masses or mass to charge
ratios, or exact masses or mass to charge ratios, may be searched
against the library of known proteins, and where more than one
protein is suggested the correct protein may be confirmed by
searching for fragment, product, daughter or adduct ions which
match those to be expected from the relevant peptides from each
candidate protein.
[0206] The step of identifying each parent or precursor ion of
interest preferably comprises recalling the elution time of the
parent or precursor ion of interest, generating a list of possible
fragment, product, daughter or adduct ions which comprises
previously recognised fragment, product, daughter or adduct ions
which are present in both the low fragmentation or reaction mass
spectrum obtained immediately before the elution time of the parent
or precursor ion of interest and the low fragmentation or reaction
mass spectrum obtained immediately after the elution time of the
parent or precursor ion of interest, generating a mass chromatogram
of each possible fragment, product, daughter or adduct ion,
determining the centre of each peak in each possible fragment,
product, daughter or adduct ion mass chromatogram, and determining
the corresponding possible fragment, product, daughter or adduct
ion elution time(s). The possible fragment, product, daughter or
adduct ions may then be ranked according to the closeness of fit of
their elution time with the elution time of the parent or precursor
ion of interest. A list of fragment, product, daughter or adduct
ions may then be formed by rejecting fragment, product, daughter or
adduct ions if the elution time of the fragment, product, daughter
or adduct ion precedes or exceeds the elution time of the parent or
precursor ion of interest by more than a predetermined amount.
[0207] The list of fragment, product, daughter or adduct ions may
be yet further refined or reduced by generating a list of
neighbouring parent or precursor ions which are present in the low
fragmentation or reaction mass spectrum obtained nearest in time to
the elution time of the final candidate parent or precursor ion. A
mass chromatogram of each parent or precursor ion contained in the
list is then generated and the centre of each mass chromatogram is
determined along with the corresponding neighbouring parent or
precursor ion elution time(s). Any fragment, product, daughter or
adduct ion having an elution time which corresponds more closely
with a neighbouring parent or precursor ion elution time than with
the elution time of a parent or precursor ion of interest may then
be rejected from the list of fragment, product, daughter or adduct
ions.
[0208] Fragment, daughter, product or adduct ions may be assigned
to a parent or precursor ion according to the closeness of fit of
their elution times, and all fragment, product, daughter or adduct
ions which have been associated with the parent or precursor ion
may be listed.
[0209] An alternative embodiment which involves a greater amount of
data processing but yet which is intrinsically simpler is also
contemplated. Once parent and fragment, product, daughter or adduct
ions have been identified, then a parent or precursor ion mass
chromatogram for each recognised parent or precursor ion is
generated. The centre of each peak in the parent or precursor ion
mass chromatogram and the corresponding parent or precursor ion
elution time(s) are then determined. Similarly, a fragment,
product, daughter or adduct ion mass chromatogram for each
recognised fragment, product, daughter or adduct ion is generated,
and the centre of each peak in the fragment, product, daughter or
adduct ion mass chromatogram and the corresponding fragment,
product, daughter or adduct ion elution time(s) are then
determined. Rather than then identifying only a sub-set of the
recognised parent or precursor ions, all (or nearly all) of the
recognised parent or precursor ions are then identified. Fragment
ions are assigned to parent or precursor ions according to the
closeness of fit of their respective elution times and all
fragment, product, daughter or adduct ions which have been
associated with a parent or precursor ion may then be listed.
[0210] Passing ions through a mass filter, preferably a quadrupole
mass filter, prior to being passed to the collision, fragmentation
or reaction device presents an alternative or an additional method
of recognising a fragment, product, daughter or adduct ion. A
fragment, product, daughter or adduct ion may be recognised by
recognising ions in a high fragmentation or reaction mass spectrum
which have a mass to charge ratio which is not transmitted by the
collision, fragmentation or reaction device i.e. fragment, product,
daughter or adduct ions are recognised by virtue of their having a
mass to charge ratio falling outside of the transmission window of
the mass filter. If the ions would not be transmitted by the mass
filter then they must have been produced in the collision,
fragmentation or reaction device.
[0211] Various embodiments of the present invention will now be
described, by way of example only, and with reference to the
accompanying drawings in which:
[0212] FIG. 1 is a schematic drawing of a preferred mass
spectrometer;
[0213] FIG. 2 shows a schematic of a valve switching arrangement
during sample loading and desalting and the inset shows desorption
of a sample from an analytical column;
[0214] FIG. 3A shows a fragment or daughter ion mass spectrum and
FIG. 3B shows the corresponding parent or precursor ion mass
spectrum obtained when a mass filter upstream of a collision cell
was arranged so as to transmit ions having a mass to charge ratio
>350 to the collision cell;
[0215] FIG. 4A shows a mass chromatogram of a parent or precursor
ion, FIG. 4B shows a mass chromatogram of a parent or precursor
ion, FIG. 4C shows a mass chromatogram of a parent or precursor
ion, FIG. 4D shows a mass chromatogram of a fragment or daughter
ion and FIG. 4E shows a mass chromatogram of a fragment or
daughter;
[0216] FIG. 5 shows the mass chromatograms of FIGS. 4A-E
superimposed upon one another;
[0217] FIG. 6 shows a mass chromatogram of the Asparagine immonium
ion which has a mass to charge ratio of 87.04;
[0218] FIG. 7 shows a mass spectrum of the peptide ion T5 derived
from ADH which has the sequence ANELLINVK and a molecular weight of
1012.59;
[0219] FIG. 8 shows a mass spectrum of a tryptic digest of
.beta.-Casein obtained when a collision cell was in a low
fragmentation mode;
[0220] FIG. 9 shows a mass spectrum of a tryptic digest of
.beta.-Casein obtained when a collision cell was in a high
fragmentation mode;
[0221] FIG. 10 shows a processed and expanded view of the mass
spectrum shown in FIG. 9;
[0222] FIG. 11A shows a mass chromatogram of an ion from a first
sample having a mass to charge ratio of 880.4, FIG. 11B shows a
similar mass chromatogram of the same ion from a second sample,
FIG. 11C shows a mass chromatogram of an ion from a first sample
having a mass to charge ratio of 582.3 and FIG. 11D shows a similar
mass chromatogram of the same ion from a second sample;
[0223] FIG. 12A shows a mass spectrum recorded from a first sample
and FIG. 12B shows a corresponding mass spectrum recorded from a
second sample which is similar to the first sample except that it
contains a higher concentration of the digest products of the
protein Casein which is common to both samples;
[0224] FIG. 13 shows the mass spectrum shown in FIG. 12A in more
detail and the insert shows an expanded part of the mass spectrum
showing isotope peaks at mass to charge ratio 880.4; and
[0225] FIG. 14 shows the mass spectrum shown in FIG. 12B in more
detail and the insert shows an expanded part of the mass spectrum
showing isotope peaks at mass to charge ratio 880.4.
[0226] A preferred embodiment will now be described with reference
to FIG. 1. A mass spectrometer 6 is shown which comprises an ion
source 1, preferably an Electrospray Ionisation source, an ion
guide 2, a quadrupole mass filter 3, a collision, fragmentation or
reaction device 4 and an orthogonal acceleration Time of Flight
mass analyser 5 incorporating a reflectron. The ion guide 2 and
mass filter 3 may be omitted if necessary. The mass spectrometer 6
is preferably interfaced with a chromatograph, such as a liquid
chromatograph (not shown) so that the sample entering the ion
source 1 may be taken from the eluent of the liquid
chromatograph.
[0227] The quadrupole mass filter 3 is preferably disposed in an
evacuated chamber which is maintained at a relatively low pressure
e.g. less than 10.sup.B5 mbar. The rod electrodes comprising the
mass filter 3 are connected to a power supply which generates both
RF and DC potentials which determine the mass to charge value
transmission window of the mass filter 3.
[0228] The collision, fragmentation or reaction device 4 may
comprise a Surface Induced Dissociation ("SID") collision,
fragmentation or reaction device, an Electron Transfer Dissociation
collision, fragmentation or reaction device, an Electron Capture
Dissociation collision, fragmentation or reaction device, an
Electron Collision or Impact Dissociation collision, fragmentation
or reaction device, a Photo Induced Dissociation ("PID") collision,
fragmentation or reaction device, a Laser Induced Dissociation
collision, fragmentation or reaction device, an infrared radiation
induced dissociation device, an ultraviolet radiation induced
dissociation device, a thermal or temperature source collision,
fragmentation or reaction device, an electric field induced
collision, fragmentation or reaction device, a magnetic field
induced collision, fragmentation or reaction device, an enzyme
digestion or enzyme degradation collision, fragmentation or
reaction device, an ion-ion reaction collision, fragmentation or
reaction device, an ion-molecule reaction collision, fragmentation
or reaction device, an ion-atom reaction collision, fragmentation
or reaction device, an ion-metastable ion reaction collision,
fragmentation or reaction device, an ion-metastable molecule
reaction collision, fragmentation or reaction device, an
ion-metastable atom reaction collision, fragmentation or reaction
device, an ion-ion reaction device for reacting ions to form adduct
or product ions, an ion-molecule reaction device for reacting ions
to form adduct or product ions, an ion-atom reaction device for
reacting ions to form adduct or product ions, an ion-metastable ion
reaction device for reacting ions to form adduct or product ions,
an ion-metastable molecule reaction device for reacting ions to
form adduct or product ions or an ion-metastable atom reaction
device for reacting ions to form adduct or product ions.
[0229] Alternatively, the collision, fragmentation or reaction
device may form part of the ion source. For example, the collision,
fragmentation or reaction device may comprise a nozzle-skimmer
interface collision, fragmentation or reaction device, an in-source
collision, fragmentation or reaction device or an ion-source
Collision Induced Dissociation collision, fragmentation or reaction
device.
[0230] In an arrangement the collision, fragmentation or reaction
device 4 may comprise either a quadrupole or hexapole rod set which
may be enclosed in a substantially gas-tight casing (other than
having a small ion entrance and exit orifice) into which a gas such
as helium, argon, nitrogen, air or methane may be introduced at a
pressure of between 10.sup.-4 and 10.sup.-1 mbar, further
preferably 10-.sup.3 mbar to 10.sup.-2 mbar. Suitable AC or RF
potentials for the electrodes comprising the collision,
fragmentation or reaction device 4 are provided by a power supply
(not shown).
[0231] Ions generated by the ion source 1 are transmitted by ion
guide 2 and pass via an interchamber orifice 7 into vacuum chamber
8. Ion guide 2 is maintained at a pressure intermediate that of the
ion source and the vacuum chamber 8. In the embodiment shown, ions
are mass filtered by mass filter 3 before entering the preferred
collision, fragmentation or reaction device 4. However, the mass
filter 3 is an optional feature of this embodiment. Ions exiting
from the collision, fragmentation or reaction device 4 or which
have been transmitted through the collision, fragmentation or
reaction device 4 preferably pass to a mass analyser which
preferably comprises a Time of Flight mass analyser 5. Other ion
optical components, such as further ion guides and/or electrostatic
lenses, may be provided which are not shown in the figures or
described herein. Such components may be used to maximise ion
transmission between various parts or stages of the apparatus.
Various vacuum pumps (not shown) may be provided for maintaining
optimal vacuum conditions. The Time of Flight mass analyser 5
incorporating a reflectron operates in a known way by measuring the
transit time of the ions comprised in a packet of ions so that
their mass to charge ratios can be determined.
[0232] A control means (not shown) provides control signals for the
various power supplies (not shown) which respectively provide the
necessary operating potentials for the ion source 1, ion guide 2,
quadrupole mass filter 3, collision, fragmentation or reaction
device 4 and the Time of Flight mass analyser 5. These control
signals determine the operating parameters of the instrument, for
example the mass to charge ratios transmitted through the mass
filter 3 and the operation of the analyser 5. The control means may
be a computer (not shown) which may also be used to process the
mass spectral data acquired. The computer can also display and
store mass spectra produced by the analyser 5 and receive and
process commands from an operator. The control means may be
automatically set to perform various methods and make various
determinations without operator intervention, or may optionally
require operator input at various stages.
[0233] The control means is also preferably arranged to switch,
alter or vary the collision, fragmentation or reaction device 4
back and forth repeatedly and/or regularly between at least two
different modes. In one mode a relatively high voltage such as
greater than or equal to 15V may be applied to the collision,
fragmentation or reaction device 4 which in combination with the
effect of various other ion optical devices upstream of the
collision, fragmentation or reaction device 4 may be sufficient to
cause a fair degree of fragmentation or reaction of ions passing
therethrough. In a second mode a relatively low voltage such as
less than or equal to 5V may be applied which may cause relatively
little (if any) significant fragmentation or reaction of ions
passing therethrough.
[0234] In one embodiment the control means may switch, alter or
vary between modes approximately every second. When the mass
spectrometer 6 is used in conjunction with an ion source 1 being
provided with an eluent separated from a mixture by means of liquid
or gas chromatography, the mass spectrometer 6 may be run for
several tens of minutes over which period of time several hundred
high and low fragmentation or reaction mass spectra may be
obtained.
[0235] At the end of the experimental run the data which has been
obtained is preferably analysed and parent or precursor ions and
fragment, product, daughter or adduct ions can be recognised on the
basis of the relative intensity of a peak in a mass spectrum
obtained when the collision, fragmentation or reaction device 4 was
in one mode compared with the intensity of the same peak in a mass
spectrum obtained approximately a second later in time when the
collision, fragmentation or reaction device 4 was in the second
mode.
[0236] According to an embodiment, mass chromatograms for each
parent and fragment, product, daughter or adduct ion are generated
and fragment, product, daughter or adduct ions are assigned to
parent or precursor ions on the basis of their relative elution
times.
[0237] An advantage of this method is that since all the data is
acquired and subsequently processed then all fragment, product,
daughter or adduct ions may be associated with a parent or
precursor ion by closeness of fit of their respective elution
times. This allows all the parent or precursor ions to be
identified from their fragment, product, daughter or adduct ions,
irrespective of whether or not they have been discovered by the
presence of a characteristic fragment, product, daughter or adduct
ion or characteristic "neutral loss".
[0238] According to another embodiment an attempt is made to reduce
the number of parent or precursor ions of interest. A list of
possible (i.e. not yet finalised) parent or precursor ions of
interest may be formed by looking for parent or precursor ions
which may have given rise to a predetermined fragment, product,
daughter or adduct ion of interest e.g. an immonium ion from a
peptide. Alternatively, a search may be made for parent and
fragment, product, daughter or adduct ions wherein the parent or
precursor ion could have fragmented or reacted into a first
component comprising a predetermined ion or neutral particle and a
second component comprising a fragment, product, daughter or adduct
ion. Various steps may then be taken to further reduce/refine the
list of possible parent or precursor ions of interest to leave a
number of parent or precursor ions of interest which are then
preferably subsequently identified by comparing elution times of
the parent or precursor ions of interest and fragment, product,
daughter or adduct ions. As will be appreciated, two ions could
have similar mass to charge ratios but different chemical
structures and hence would most likely fragment differently
enabling a parent or precursor ion to be identified on the basis of
a fragment, product, daughter or adduct ion.
[0239] A sample introduction system is shown in more detail in FIG.
2. Samples may be introduced into the mass spectrometer 6 by means
of a Micromass (RTM) modular CapLC system. For example, samples may
be loaded onto a C18 cartridge (0.3 mm.times.5 mm) and desalted
with 0.1% HCOOH for 3 minutes at a flow rate of 30 uL per minute. A
ten port valve may then switched such that the peptides are eluted
onto the analytical column for separation, see inset of FIG. 2.
Flow from two pumps A and B may be split to produce a flow rate
through the column of approximately 200 nl/min.
[0240] A preferred analytical column is a PicoFrit (RTM) column
packed with Waters (RTM) Symmetry C18 set up to spray directly into
the mass spectrometer 6. An Electrospray potential (ca. 3kV) may be
applied to the liquid via a low dead volume stainless steel union.
A small amount e.g. 5 psi (34.48 kPa) of nebulising gas may be
introduced around the spray tip to aid the Electrospray
process.
[0241] Data can be acquired using a mass spectrometer 6 fitted with
a Z-spray (RTM) nanoflow Electrospray ion source. The mass
spectrometer may be operated in the positive ion mode with a source
temperature of 80.degree. C. and a cone gas flow rate of
401/hr.
[0242] The instrument may be calibrated with a multi-point
calibration using selected fragment, product, daughter or adduct
ions that result, for example, from the Collision Induced
Decomposition (CID) of Glu-fibrinopeptide b. Data may be processed
using the MassLynx (RTM) suite of software.
[0243] FIGS. 3A and 3B show respectively fragment or daughter and
parent or precursor ion spectra of a tryptic digest of alcohol
dehydrogenase (ADH). The fragment or daughter ion spectrum shown in
FIG. 3A was obtained while the collision cell voltage was high,
e.g. around 30V, which resulted in significant fragmentation of
ions passing therethrough. The parent or precursor ion spectrum
shown in FIG. 3B was obtained at low collision energy e.g. less
than or equal to 5V. The data presented in FIG. 3B was obtained
using a mass filter 3 upstream of the collision cell and set to
transmit ions having a mass to charge value greater than 350. The
mass spectra in this particular example were obtained from a sample
eluting from a liquid chromatograph, and the spectra were obtained
sufficiently rapidly and close together in time that they
essentially correspond to the same component or components eluting
from the liquid chromatograph.
[0244] In FIG. 3B, there are several high intensity peaks in the
parent or precursor ion spectrum, e.g. the peaks at 418.7724 and
568.7813, which are substantially less intense in the corresponding
fragment or daughter ion spectrum shown in FIG. 3A. These peaks may
therefore be recognised as being parent or precursor ions.
Likewise, ions which are more intense in the fragment or daughter
ion spectrum shown in FIG. 3A than in the parent or precursor ion
spectrum shown in FIG. 3B may be recognised as being fragment or
daughter ions. As will also be apparent, all the ions having a mass
to charge value less than 350 in the high fragmentation mass
spectrum shown in FIG. 3A can be readily recognised as being
fragment or daughter ions on the basis that they have a mass to
charge value less than 350 and the fact that only parent or
precursor ions having a mass to charge value greater than 350 were
transmitted by the mass filter 5 to the collision cell.
[0245] FIGS. 4A-E show respectively mass chromatograms for three
parent or precursor ions and two fragment or daughter ions. The
parent or precursor ions were determined to have mass to charge
ratios of 406.2 (peak "MC1"), 418.7 (peak "MC2") and 568.8 (peak
"MC3") and the two fragment or daughter ions were determined to
have mass to charge ratios of 136.1 (peaks "MC4" and "MC5") and
120.1 (peak "MC6").
[0246] It can be seen that parent or precursor ion peak MC1 (mass
to charge ratio 406.2) correlates well with fragment or daughter
ion peak MC5 (mass to charge ratio 136.1) i.e. a parent or
precursor ion with a mass to charge ratio of 406.2 seems to have
fragmented to produce a fragment or daughter ion with a mass to
charge ratio of 136.1. Similarly, parent or precursor ion peaks MC2
and MC3 correlate well with fragment or daughter ion peaks MC4 and
MC6, but it is difficult to determine which parent or precursor ion
corresponds with which fragment or daughter ion.
[0247] FIG. 5 shows the peaks of FIGS. 4-E overlaid on top of one
other and redrawn at a different scale. By careful comparison of
the peaks of MC2, MC3, MC4 and MC6 it can be seen that in fact
parent or precursor ion MC2 and fragment or daughter ion MC4
correlate well whereas parent or precursor ion MC3 correlates well
with fragment or daughter ion MC6. This suggests that parent or
precursor ions with a mass to charge ratio of 418.7 fragmented to
produce fragment or daughter ions with a mass to charge ratio of
136.1 and that parent or precursor ions with mass to charge ratio
568.8 fragmented to produce fragment or daughter ions with a mass
to charge ratio of 120.1.
[0248] This cross-correlation of mass chromatograms may be carried
out using automatic peak comparison means such as a suitable peak
comparison software program running on a suitable computer.
[0249] FIG. 6 show the mass chromatogram for the fragment or
daughter ion having a mass to charge ratio of 87.04 extracted from
a HPLC separation and mass analysis obtained using mass
spectrometer 6. It is known that the immonium ion for the amino
acid Asparagine has a mass to charge value of 87.04. This
chromatogram was extracted from all the high energy spectra
recorded on the mass spectrometer 6. FIG. 7 shows the full mass
spectrum corresponding to scan number 604. This was a low energy
mass spectrum recorded on the mass spectrometer 6, and is the low
energy spectrum next to the high energy spectrum at scan 605 that
corresponds to the largest peak in the mass chromatogram of mass to
charge ratio 87.04. This shows that the parent or precursor ion for
the Asparagine immonium ion at mass to charge ratio 87.04 has a
mass of 1012.54 since it shows the singly charged (M+H).sup.+ ion
at mass to charge ratio 1013.54, and the doubly charged
(M+2H).sup.++ ion at mass to charge ratio 507.27.
[0250] FIG. 8 shows a mass spectrum from a low energy spectra
recorded on a mass spectrometer 6 of a tryptic digest of the
protein .beta.-Casein. The protein digest products were separated
by HPLC and mass analysed. The mass spectra were recorded on a mass
spectrometer 6 operating in a MS mode and alternating between low
and high collision energy in a gas collision cell for successive
spectra. FIG. 9 shows a mass spectrum from the high energy spectra
recorded at substantially the same time that the low energy mass
spectrum shown in FIG. 8 relates to. FIG. 10 shows a processed and
expanded view of the mass spectrum shown in FIG. 9 above. For this
spectrum, the continuum data has been processed so as to identify
peaks and display them as lines with heights proportional to the
peak area, and annotated with masses corresponding to their
centroided masses. The peak at mass to charge ratio 1031.4395 is
the doubly charged (M+2H).sup.++ ion of a peptide, and the peak at
mass to charge ratio 982.4515 is a doubly charged fragment or
daughter ion. It has to be a fragment or daughter ion since it is
not present in the low energy spectrum. The mass difference between
these ions is 48.9880. The theoretical mass for H.sub.3PO.sub.4 is
97.9769, and the mass to charge value for the doubly charged
H.sub.3PO.sub.4.sup.++ ion is 48.9884, a difference of only 8 ppm
from that observed. It is therefore assumed that the peak having a
mass to charge ratio of 982.4515 relates to a fragment or daughter
ion resulting from a peptide ion having a mass to charge of
1031.4395 losing a H.sub.3PO.sub.4.sup.++ ion.
[0251] Some experimental data is now presented which illustrates
the ability of the preferred embodiment to quantify the relative
abundance of two proteins contained in two different samples which
comprise a mixture of proteins.
[0252] A first sample contained the tryptic digest products of
three proteins BSA, Glycogen Phosphorylase B and Casein. These
three proteins were initially present in the ratio 1:1:1. Each of
the three proteins had a concentration of 330 fmol/ul. A second
sample contained the tryptic digest products of the same three
proteins BSA, Glycogen Phosphorylase B and Casein. However, the
proteins were initially present in the ratio 1:1:X. X was uncertain
but believed to be in the range 2-3. The concentration of the
proteins BSA and Glycogen Phosphorylase B in the second sample
mixture was the same as in the first sample, namely 330
fmol/.mu.l.
[0253] The experimental protocol which was followed was that 1
.mu.l of sample was loaded for separation on to a HPLC column at a
flow rate of 4 .mu.l/min. The liquid flow was then split such that
the flow rate to the nano-electrospray ionisation source was
approximately 200 nl/min.
[0254] Mass spectra were recorded on the mass spectrometer 6. Mass
spectra were recorded at alternating low and high collision energy
using nitrogen collision gas. The low-collision energy mass spectra
were recorded at a collision voltage of 10V and the high-collision
energy mass spectra were recorded at a collision voltage of 33V.
The mass spectrometer was fitted with a Nano-Lock-Spray device
which delivered a separate liquid flow to the source which may be
occasionally sampled to provide a reference mass from which the
mass calibration may be periodically validated. This ensured that
the mass measurements were accurate to within an RMS accuracy of 5
ppm. Data were recorded and processed using the MassLynx (RTM) data
system.
[0255] The first sample was initially analysed and the data was
used as a reference. The first sample was then analysed a further
two times. The second sample was analysed twice. The data from
these analyses were used to attempt to quantify the (unknown)
relative abundance of Casein in the second sample.
[0256] All data files were processed automatically generating a
list of ions with associated areas and high-collision energy
spectra for each experiment. This list was then searched against
the Swiss-Prot protein database using the ProteinLynx (RTM) search
engine. Chromatographic peak areas were obtained using the Waters
(RTM) Apex Peak Tracking algorithm. Chromatograms for each charge
state found to be present were summed prior to integration.
[0257] The experimentally determined relative expression level of
various peptide ions normalised with respect to the reference data
for the two samples are given in the following tables.
TABLE-US-00001 BSA peptide Sample 1 Sample 1 Sample 2 Sample 2 ions
Run 1 Run 2 Run 1 Run 2 FKDLGEEHFK 0.652 0.433 0.914 0.661
HLVDEPQNLTK 0.905 0.829 0.641 0.519 KVPQVSTPTLVEVSR 1.162 0.787
0.629 0.635 LVNELTEFAK 1.049 0.795 0.705 0.813 LGEYGFQNALIVR 1.278
0.818 0.753 0.753 AEFVEVTK 1.120 0.821 0.834 0.711 Average 1.028
0.747 0.746 0.682
[0258] TABLE-US-00002 Glycogen Phophorylase B Sample 1 Sample 1
Sample 2 Sample 2 peptide ions Run 1 Run 2 Run 1 Run 2 VLVDLER
1.279 0.751 n/a 0.701 TNFDAFPDK 0.798 0.972 0.691 0.699 EIWGVEPSR
0.734 0.984 1.053 1.054 LITAIGDVVNHDPVVGDR 1.043 0.704 0.833 0.833
VLPNDNFFEGK 0.969 0.864 0.933 0.808 QIIEQLSSGFFSPK 0.691 n/a 1.428
1.428 VAAAFPGDVDR 1.140 0.739 0.631 0.641 Average 0.951 0.836 0.928
0.881
[0259] TABLE-US-00003 CASEIN Peptide Sample 1 Sample 1 Sample 2
Sample 2 sequence Run 1 Run 2 Run 1 Run 2 EDVPSER 0.962 0.941 2.198
1.962 HQGLPQEVLNENLLR 0.828 0.701 1.736 2.090 FFVAPFPEVFGK 1.231
0.849 2.175 1.596 Average 1.007 0.830 2.036 1.883
[0260] Peptides whose sequences were confirmed by high-collision
energy data are underlined in the above tables. Confirmation means
that the probability of this peptide, given its accurate mass and
the corresponding high-collision energy data, is larger than that
of any other peptide in the database given the current
fragmentation or reaction model. The remaining peptides are
believed to be correct based on their retention time and mass
compared to those for confirmed peptides. It was expected that
there would be some experimental error in the results due to
injection volume errors and other effects.
[0261] When using BSA as an internal reference, the relative
abundance of Glycogen Phosphorylase B in the first sample was
determined to be 0.925 (first analysis) and 1.119 (second analysis)
giving an average of 1.0. The relative abundance of Glycogen
Phosphorylase B in the second sample was determined to be 1.244
(first analysis) and 1.292 (second analysis) giving an average of
1.3. These results compare favourably with the expected value of
1.
[0262] Similarly, the relative abundance of Casein in the first
sample was determined to be 0.980 (first analysis) and 1.111
(second analysis) giving an average of 1.0. The relative abundance
of Casein in the second sample was determined to be 2.729 (first
analysis) and 2.761 (second analysis) giving an average of 2.7.
These results compare favourably with the expected values of 1 and
2-3.
[0263] The following data relates to chromatograms and mass spectra
obtained from the first and second samples. One peptide having the
sequence HQGLPQEVLNENLLR and derived from Casein elutes at almost
exactly the same time as the peptide having the sequence LVNELTEFAK
derived from BSA. Although this is an unusual occurrence, it
provided an opportunity to compare the abundance of Casein in the
two different samples.
[0264] FIGS. 11A-D show four mass chromatograms, two relating to
the first sample and two relating to the second sample. FIG. 11A
shows a mass chromatogram relating to the first sample for ions
having a mass to charge ratio of 880.4 which corresponds with the
peptide ion (M+2H).sup.++ having the sequence HQGLPQEVLNENLLR and
which is derived from Casein. FIG. 11B shows a mass chromatogram
relating to the second sample which corresponds with the same
peptide ion having the sequence HQGLPQEVLNENLLR which is derived
from Casein.
[0265] FIG. 11C shows a mass chromatogram relating to the first
sample for ions having a mass to charge ratio of 582.3 which
corresponds with the peptide ion (M+2H).sup.++ having the sequence
LVNELTEFAK and which is derived from BSA. FIG. 11D shows a mass
chromatogram relating to the second sample which corresponds with
the same peptide ion having the sequence LVNELTEFAK and which is
derived from BSA. The mass chromatograms show that the peptide ions
having a mass to charge ratio of mass to charge ratio 582.3 derived
from BSA are present in both samples in roughly equal amounts
whereas there is approximately a 100% difference in the intensity
of peptide ion having a mass to charge ratio of 880.4 derived from
Casein.
[0266] FIG. 12A show a parent or precursor ion mass spectrum
recorded after around 20 minutes from the first sample and FIG. 12B
shows a parent or precursor ion mass spectrum recorded after around
substantially the same time from the second sample. The mass
spectra show that the ions having a mass to charge ratio of 582.3
(derived from BSA) are approximately the same intensity in both
mass spectra whereas ions having a mass to charge ratio of 880.4
which relate to a peptide ion from Casein are approximately twice
the intensity in the second sample compared with the first sample.
This is consistent with expectations.
[0267] FIG. 13 shows the parent or precursor ion mass spectrum
shown in FIG. 12A in more detail. Peaks corresponding with BSA
peptide ions having a mass to charge of 582.3 and peaks
corresponding with the Casein peptide ions having a mass to charge
ratio of 880.4 can be clearly seen. The insert shows the expanded
part of the spectrum showing the isotope peaks of the peptide ion
having a mass to charge ratio of 880.4. Similarly, FIG. 14 shows
the parent or precursor ion mass spectrum shown in FIG. 12B in more
detail. Again, peaks corresponding with BSA peptide ions having a
mass to charge ratio of 582.3 and peaks corresponding with the
Casein peptide ions having a mass to charge ratio of 880.4 can be
clearly seen. The insert shows the expanded part of the spectrum
showing the isotope peaks of the peptide ion having a mass to
charge ratio of 880.4. It is apparent from FIGS. 12-14 and from
comparing the inserts of FIGS. 13 and 14 that the abundance of the
peptide ion derived from Casein which has a mass spectral peak of
mass to charge ratio 880.4 is approximately twice the abundance in
the second sample compared with the first sample.
[0268] Although the present invention has been described with
reference to preferred embodiments, it will be understood by those
skilled in the art that various changes in form and detail may be
made without departing from the scope of the invention as set forth
in the accompanying claims.
* * * * *