U.S. patent application number 12/791435 was filed with the patent office on 2011-01-06 for specific analysis of analytes using reagent compounds, labeling strategies, and mass spectrometry workflow.
This patent application is currently assigned to DH TECHNOLOGIES DEVELOPMENT PTE. LTD.. Invention is credited to Subhakar DEY, Sasi PILLAI, Subhasish PURKAYASTHA, Michal Weinstock, Brian L. WILLIAMSON.
Application Number | 20110003395 12/791435 |
Document ID | / |
Family ID | 43298006 |
Filed Date | 2011-01-06 |
United States Patent
Application |
20110003395 |
Kind Code |
A1 |
DEY; Subhakar ; et
al. |
January 6, 2011 |
SPECIFIC ANALYSIS OF ANALYTES USING REAGENT COMPOUNDS, LABELING
STRATEGIES, AND MASS SPECTROMETRY WORKFLOW
Abstract
Labeling reagents, sets of labeling reagents, and labeling
techniques are provided for the relative quantitation, absolute
quantitation, or both, of ketone or aldehyde compounds including,
but not limited to, analytes comprising steroids or ketosteroids.
The analytes can be medical or pharmaceutical compounds in
biological samples. Methods for labeling, analyzing, and
quantifying ketone or aldehyde compounds are also disclosed as are
methods that also use mass spectrometry.
Inventors: |
DEY; Subhakar; (N.
Billerica, MA) ; PILLAI; Sasi; (Littleton, MA)
; WILLIAMSON; Brian L.; (Ashland, MA) ;
PURKAYASTHA; Subhasish; (Acton, MA) ; Weinstock;
Michal; (Newton, MA) |
Correspondence
Address: |
KILYK & BOWERSOX, P.L.L.C.
3925 CHAIN BRIDGE ROAD, SUITE D401
FAIRFAX
VA
22030
US
|
Assignee: |
DH TECHNOLOGIES DEVELOPMENT PTE.
LTD.
Singapore
SG
|
Family ID: |
43298006 |
Appl. No.: |
12/791435 |
Filed: |
June 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61182748 |
May 31, 2009 |
|
|
|
Current U.S.
Class: |
436/98 ;
544/400 |
Current CPC
Class: |
G01N 33/64 20130101;
H01J 49/00 20130101; Y10T 436/147777 20150115; G01N 33/743
20130101; G01N 27/62 20130101; H01J 49/164 20130101; G01N 2030/045
20130101; G01N 2030/884 20130101; H01J 49/0045 20130101; G01N 30/72
20130101; Y10T 436/200833 20150115 |
Class at
Publication: |
436/98 ;
544/400 |
International
Class: |
G01N 33/00 20060101
G01N033/00; C07D 241/04 20060101 C07D241/04 |
Claims
1. A set of mass labels comprising two or more compounds of the
general formula (II): Z--R.sub.1, wherein one or more of the
compounds in the set of labels contains one or more heavy atom
isotopes and wherein, Z represents a mass reporter group comprised
of substituted or unsubstituted straight, branched or cyclic alkyl;
a substituted or unsubstituted aryl; a substituted or unsubstituted
hetero aryl; a substituted or unsubstituted amino; or a substituted
or unsubstituted thio; and R.sub.1 represents a terminal aminoxy
group.
2. The set of labels of claim 1, wherein R.sub.1 is selected from
the group consisting of ##STR00012##
3. The set of labels of claim 1, wherein one or more of the
compounds of the general formula (II) of the set of labels is
isotopically enriched with two or more heavy atoms.
4. The set of labels of claim 1, wherein one or more of the
compounds of the general formula (II) of the set of labels is
isotopically enriched with three or more heavy atoms.
5. The set of labels of claim 1, wherein the heavy atom isotopes
are each independently .sup.13C, .sup.15N, .sup.18O, .sup.33S, or
.sup.34S.
6. The set of labels of claim 1, wherein the labels are isobaric in
the unsalted or unhydrated form, and each of the isobaric labels
contains one or more heavy atom isotopes.
7. The set of labels of claim 1, wherein each label in the set of
labels being differentiated from the another label in the set by a
mass difference greater than about 1 amu.
8. The set of labels of claim 1, wherein at least one of the two or
more compounds is a compound of the formula:
Y--(CH.sub.2)n-ONH.sub.2 wherein n is an integer from 1 to 100 and
Y is selected from the group consisting of: ##STR00013##
9. The set of labels of claim 8, wherein n is an integer from 2 to
10.
10. The set of labels of claim 1, wherein each of the two or more
compounds individually comprises a permanently charged aminoxy
reagent.
11. A kit comprising the set of mass labels of claim 1, and one or
more other assay components.
12. The kit of claim 11, wherein the one or more other assay
components comprises a buffer, a reagent, a separation column, and
instructions for carrying out an assay.
13. A method for mass analysis of an analyte in a sample
comprising: derivatizing an analyte comprising an aldehyde or
ketone functional group, with a labeling reagent comprising a
terminal aminoxy moiety and a reporter group, to form a labeled
analyte; subjecting the labeled analyte to ionization; and
detecting the analyte by mass analysis.
14. The method of claim 13, further comprising determining a
concentration of the analyte in a sample.
15. The method of claim 13, wherein the labeling reagent comprises
a permanently charged aminoxy reagent.
16. The method of claim 13, wherein the labeling reagent comprises
a compound of the formula: Y--(CH.sub.2)n-ONH.sub.2 wherein n is an
integer from 1 to 100 and Y is selected from the group consisting
of: ##STR00014##
17. The method of claim 16, wherein n is an integer from 2 to
10.
18. The method of claim 13, wherein the labeling reagent is
selected from the group consisting of ##STR00015## and combinations
thereof.
19. The method of claim 13, further comprising the step of
subjecting the labeled analyte to ion fragmentation to yield an
ionized reporter group.
20. The method of claim 13, wherein the step of detecting the
analyte is comprised of detecting ions of the reporter group in a
mass analyzer.
21. The method of claim 13, wherein the step of detecting is
comprised of detecting a first transmitted parent ion and a
daughter ion fragment by parent-daughter ion transition
monitoring.
22. The method of claim 13, further comprising derivatizing an
aldehyde or ketone functional group of a standard compound and
measuring the relative concentration of the analyte.
23. The method of claim 13, wherein the labeling reagent is
isotopically enriched with two or more heavy atoms.
24. The method of claim 13, wherein at least two analyte compounds
are derivatized with the labeling reagent and the method further
comprises the step of determining a relative concentration between
at least two analytes.
25. The method of claim 13, wherein at least two analyte compounds
are derivatized with the labeling reagent and the method further
comprises the step of determining an absolute concentration of at
least one analyte.
26. The method of claim 13, wherein the ionization produces
structurally specific fragment ions and Q3 MRM ions, the labeling
reagent is wholly or partly contained in the structurally specific
fragment ions, and the method provides both sensitivity and
specificity for the Q3 MRM ions.
27. The method of claim 8, wherein the labeling reagent is
isotopically enriched with two or more heavy atoms.
28. The method of claim 13, further comprising subjecting the
labeled analyte to liquid chromatographic separation prior to the
ionization.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of the earlier
filing date of U.S. Provisional Patent Application No. 61/182,748,
filed May 31, 2009, which is incorporated herein in its entirety by
reference.
BACKGROUND
[0002] Ketones and aldehydes are polar chemical functionalities
having a carbonyl group linked to one or two other carbon atoms.
Ketone and aldehydes compounds play an important role in industry,
agriculture, and medicine. Ketones and aldehydes are also important
agents in human metabolism and biochemistry. Ketosteroids, in
particular, are a class of ketone-containing steroid compounds and
are uniquely valuable in research and clinical diagnosis because
these compounds are critical agents in hormone-regulated biological
processes and have strong biological activity at very low
concentrations. Many ketosteroids are also potentially valuable
pharmaceutical agents and the analysis of their function and
metabolism in the body are useful in both medical treatments and
diagnostic techniques for the detection of disease.
[0003] Analysis and measurement of ketone and aldehyde compounds is
challenging because these compounds can be present at low levels in
clinical and biological samples such as plasma. Standard
chromatographic techniques such as GC-MS methods for analysis after
chemical derivatization are available but the chemical
derivatization is not specific. See, e.g., Song, J. et al., Journal
of Chromatography B., Vol. 791, Issues 1-2, (127-135) 2003. Methods
using fluorescence detection are available and some specific
immunoassays, including radioimmunoassays (RAs), are available, but
these usually do not offer multi-component analysis. The major
problems with RAs are lack of specificity and the need to perform a
different assay for each steroid.
[0004] Examples in the literature of LC/MS strategies exploit
derivatization of the analytes, but the ionization efficiency is
relatively low and these strategies have failed to achieve the
limit of detection required for the assay to be viable in a
clinical setting using mass spectrometry and also lack multiplexing
capability. Steroid analysis in biological samples is also crucial
for the evaluation and clinical detection of various endocrine and
metabolic disorders. Clinical laboratories are currently performing
radioimmunoassays (RAs) for high throughput screening of
steroids.
[0005] The above challenges posed by attempting to measure ketone
and aldehyde compounds in samples are also magnified by the desire
to rapidly screen and/or analyze a large number of biological
samples for the specific compounds of interest or a panel of ketone
or aldehyde analytes. Although mass spectrometry can provide rapid
throughput, ketone and aldehyde steroids are particularly
challenging because of interference in the mass measurements by
competing compounds and low sample concentrations in the sample
medium. In addition, some classes of ketone and aldehyde compounds,
and particularly ketosteroids, are not compatible with traditional
sample processing conditions often used to prepare samples for mass
spectrometric analysis. Ketosteroids are also particularly
challenging due to poor ionization efficiency and complex
ionization patterns during MS/MS analysis.
[0006] Therefore, although techniques for rapid and efficient
analysis and quantitation of ketone and aldehyde compounds are
highly desirable because of the biological importance of these
compounds, the existing techniques are not ideal due to lack of
sensitivity, cross-reacting substances, and other challenges
inherent in the chemistry of the compounds.
[0007] Sensitive, selective, and accurate analysis of ketosteriods
can be used for the monitoring of abnormal adrenal functions. The
ionization efficiency of native ketosteriods in positive MS/MS can
be poor, resulting often times in insufficient limits of detection
(LODs), especially when analyzing human samples from infants and
children. Derivatization of ketosteroids via their keto
functionality to form hydrazines has been used to improve
ionization and enhance sensitivity, as described, for example, in
Kushnir et al., Performance Characteristics of a Novel Tandem Mass
Spectrometry Assay For Serum Testosterone, Clin Chem. 52:1,
120-128, 2006, which is incorporated herein in its entirety by
reference.
[0008] MRM analysis and MS/MS conditions work well in clean
solvent, however, when using complex biological samples, a high
background (BKG) noise, often from the same mass Q1/Q3 interfaces,
is produced, complicating chromatography and reducing detection
limits. A need exits for a method to quantitate ketosteroids and
analytes containing a keto or aldehyde functionality.
SUMMARY
[0009] The present teachings relates to compounds, methods, and
strategies for the analysis of aldehydes and ketones, specifically
ketosteroids, in a sample. Labeling compounds are specially
designed to derivatize the ketone or aldehyde functionality of an
analyte using simple chemistry that can be applied to these
compounds in many important biological samples. The derivitization
converts a ketone or aldehyde group to an oxime, thereby imparting
a more hydrophilic nature to the analyte. Specifically, the ketone
or aldehyde functional group is derivatized using aminoxy chemistry
to create a labeled analyte that is suitable for ionization and
detection by mass spectrometry. In some embodiments, the label
reagent is comprised of a mass reporter and an aminoxy group such
that the ionized reporter group is detectable. In others, the label
comprises a neutral loss group and an aminoxy group such that the
charged analyte is detectable by mass spectrometry. In either case,
mass analysis of such labeled analytes yield improved detection
characteristics, specifically including a large increase in
selectivity and a large increase (10-1000 fold) in sensitivity of
detection. This strategy also overcomes many of the challenges
inherent in measuring or detecting ketone and aldehyde compounds in
a sample matrix. The labels and labeling strategy also result in
exclusively protonated molecular ions and fragmentation in mass
analysis yields a simplified resulting MS spectra.
[0010] The methods described herein can measure relative
concentration, absolute concentration, or both, and can be applied
to one or more steroids in one or more samples. The present methods
also include isobaric labeling reagents and methods, as well as
mass differential labeling reagents and methods, depending on the
selection of isotopic substitution and labeling strategies for the
compounds. Isotopically enriched analogues of the labeling regent
can be used and internal standards can be generated for
quantitation. United States Patent Application Publication No. US
2005/068446 A1 discloses synthesis of isotopically enriched
compounded; mass analysis workflows and strategies are disclosed in
U.S. Patent Application Publication No. US 2008/0014642 A1, both of
which are incorporated herein in their entireties by reference.
[0011] The present teachings provide a method for quantifying
ketosteroids and analytes containing keto or aldehyde
functionality. In some embodiments, the method can comprise
derivatization chemistry and a liquid chromatography/tandem mass
spectrometry (LC/MSMS) workflow. The method can comprise using a
permanently charged aminoxy reagent which significantly increases
the detection limits of ketosteroids. Exemplary aminoxy reagents
that can be used include those of formula (I):
Y--(CH.sub.2)n-ONH.sub.2 (I)
wherein n is an integer from 1 to 100 and Y can be any one of these
moieties:
##STR00001##
In some embodiments, n is an integer from 2 to 50, or from 2 to 20,
or from 2 to 10, or from 2 to 6, or from 3 to 8. In some
embodiments, n can be 3 or 4. In some embodiments, Y can be a
different charged moiety than any of these four. Y can be a
permanently charged moiety, for example, a permanently charged
phosphorus-containing or nitrogen-containing moiety. In some
embodiments, a kit including one or more of the aminoxy reagents
described herein, can be provided.
[0012] The method can involve using an MRM workflow for
quantitative analysis of ketosteroids. The reagents can be
isotope-coded for quantitative analysis of an individual or of a
panel of keto compounds. The MS/MS fragmentation at low collision
energies is very clean resulting in one predominant signature ion.
The signature ion can result from a neutral loss from the
aminoxy-derivatized product. The MRM transition can be the mass of
the derivatized steroid in Q1 and the mass of the neutral loss
fragment in Q3. The present teachings provide a process for
significantly reducing background noise via derivatization,
resulting in improved sensitivity and targeted selection of Q3
fragments resulting in improved specificity.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0013] FIG. 1 is a schematic diagram of a one step derivatization
reaction of four ketosteroid compounds using labeling reagents
disclosed herein.
[0014] FIG. 2 shows four chemical structures of four mass
differential reagents for derivatizing a ketone or aldehyde
analyte.
[0015] FIGS. 3A-3D are MS spectrum of derivatized and underivatized
ketosteroid analytes: 3A is underivatized (top) and derivatized
(bottom) testosterone; 3B is underivatized (top) and derivatized
(bottom) progesterone; 3C is underivatized (top) and derivatized
(bottom) pregnenolone; and 3D is underivatized (top) and
derivatized (bottom) aldosterone.
[0016] FIG. 4 is triplicate MALDI-MRM peaks for derivatized (1
pg/spot) and underivatized (48 pg/spot) aldosterone, testosterone,
and pregnenolone.
[0017] FIG. 5 is concentration calibration curves for testosterone,
aldosterone, and pregnenolone.
[0018] FIG. 6 is chemical structures of suitable terminal aminoxy
groups suitable for the labeling reagents disclosed herein and a
quarternary amino mass balance moiety also comprised at a terminal
aminoxy functionality.
[0019] FIG. 7 is an outline of an analytical strategy and workflow
method for derivatizing, analyzing, and calculating the
concentration of a ketone or aldehyde compound using the techniques
described herein.
[0020] FIGS. 8A-8B are MS spectra of underivatized (8A) and
derivatized (8B) cortexolone. FIG. 8A shows a complex fragmentation
patterns across the entire spectrum while FIG. 8B shows a
simplified spectrum with a strong signal from a derivatized analyte
at 117 Da.
[0021] FIG. 9 is an MALDI analysis of 1 pg of
testosterone/epitestosterone in derivatized (a) and underivatized
(b) forms.
[0022] FIG. 10 is a graph of reaction efficiency calculated to be
>99%.
[0023] FIG. 11 shows the reaction quantitation of the aminoxy
chemical derivatization in a calibration curve generated from a
serial dilution of derivatized and underivatized testosterone.
[0024] FIG. 12 shows the MS/MS fragments and spectrum of QAO
Testosterone using CE=62 eV at which the signature ions contain
fragments from both testosterone structure and from the
derivatizing reagent structure, according to various embodiments of
the present teachings.
[0025] FIG. 13 shows the chromatograms of QAO derivatized
testosterone using and MRM transition of a targeted Q3 fragment as
compared to neutral loss Q3 fragment, according to various
embodiments of the present teachings.
[0026] FIG. 14 shows the targeted MS/MS fragmentation and spectrum
of QAO Progesterone and the MS/MS spectrum of QAO Testosterone at
CE=62 eV.
[0027] FIG. 15 shows the MS/MS spectrum of progesterone at CE=45 eV
and illustrates a background noise reduction in an LC-MS/MS
analysis.
DETAILED DESCRIPTION OF INVENTION
[0028] The ketone and aldehyde compounds used as analytes in the
mass spectrometry techniques described herein are found in a
variety of sources such as physiological fluid samples, cell or
tissue lysate samples, protein samples, cell culture samples,
agricultural product samples and essentially any sample where the
ketone and aldehyde functionality is present in the analyte. To
demonstrate the applicability of the present techniques to ketone
and aldehyde compounds, ketosteroids are analyzed and measured in
the Examples below. The ketosteroids present a particular challenge
due to the low concentrations in the matrix of common clinical
samples and the techniques, label reagents and methods applicable
thereto are readily applied to ketone or aldehyde compounds.
[0029] Moreover, the present teachings can be applied to both
natural and synthetic ketone or aldehyde analytes. Ketosteroids
including, but not limited to, DHT, testosterone, epitestosterone,
desoxymethyltestosterone (DMT), tetrahydrogestrinone (THG),
aldosterone, estrone, 4-hydroxyestrone, 2-methoxyestrone,
2-hydroxyestrone, 16-ketoestradiol, 16 alpha-hydroxyestrone,
2-hydroxyestrone-3-methylether, prednisone, prednisolone,
pregnenolone, progesterone, DHEA (dehydroepiandrosterone), 17 OH
pregnenolone, 17 OH progesterone, 17 OH progesterone, androsterone,
epiandrosterone, and D4A (delta 4 androstenedione), and can be
analyzed in various embodiments of the present teachings.
[0030] The present invention includes reagents and methods using
mass differential tags including sets of mass differential labels
where one or more labels of the set contains one or more heavy atom
isotopes. A set of mass differential labels can also be provided by
preparing labels with different overall mass and different primary
reporter ion masses or mass balance groups, although not every
member of a set of mass differential tags need be isotopically
enriched. The present reagents and methods enable analysis of
ketone and aldehyde analytes in one or more samples using mass
differential labels and parent-daughter ion transition monitoring
(PDITM). The present teachings can be used for qualitative and
quantitative analysis of such analytes using mass differential
tagging reagents and mass spectrometry. The mass differential tags
include, but are not limited to, non-isobaric isotope coded
reagents and the present invention includes reagents and methods
for the absolute quantitation of ketone and aldehyde compounds with
or without the use of an isotopically enriched standard
compound.
[0031] When isotopically enriched isobaric tags are used, sets of
isobaric labels may comprise one, or more heavy atom isotopes. A
set of isobaric labels can have an identical or specifically
defined range of aggregate masses but has a primary reporter ion or
charged analyte of a different measurable mass. A set of isobaric
reagents enables both qualitative and quantitative analysis of
ketone and aldehyde analyte compounds using mass spectroscopy. For
example, isotopically enriched isobaric tags and parent-daughter
ion transition monitoring (PDITM) can measure or detect one or more
ketone or aldehyde compounds in a sample such as a specific
ketosteroid or group of ketosteroids.
[0032] The present invention also includes kits of labeling
reagents and sets of labeling reagents for the relative
quantitation, absolute quantitation, or both, of ketone compounds
in biological samples including labeling reagents can be
represented by general formula (II):
Z--R.sub.1, (II)
and can be provided and/or used in a salt or hydrate form. In
general, in formula (II): (a) Z represents a mass reporter group
comprised of (i) a substituted or unsubstituted straight, branched
or cyclic alkyl; a substituted or unsubstituted aryl; a substituted
or unsubstituted hetero aryl; a substituted or unsubstituted amino;
or a substituted or unsubstituted thio or (ii) a quarternary
nitrogen as an amino group; and (b) R.sub.1 represents a
substituted or unsubstituted terminal aminoxy having the formula
O--NH.sub.2.
[0033] In various aspects, the present teachings provide labeled
analytes, wherein the analyte is comprised at least one ketone
group and a label described herein. The labeled ketone compounds
can be represented by the general configuration (III):
Analyte-Oxime-Label, which may be represented by the formula (III):
A-X--R. A represents the compound that contained one or more ketone
or aldehyde groups prior to formation of the labeled compound; X
represents an oxime group; and R is the label described above.
[0034] As noted above, the present teachings are not limited to the
analysis of ketosteroids, but can be applied to any compound
containing a ketone or aldehyde group by reaction of the ketone or
aldehyde group with a label or tag comprised of a terminal aminoxy
group, to yield the resulting oxime, and a reporter group or
charged analyte susceptible to detection by mass analysis.
[0035] In various embodiments, the labeling reagent or labeled
analyte compound comprises substituted or unsubstituted terminal
aminoxy as follows:
##STR00002##
including the following species
##STR00003##
[0036] The present teachings can provide reagents and methods for
the analysis of one or more ketone or aldehyde compound in one or
more samples using mass differential labels, isobaric labels, or
both, and parent-daughter ion transition monitoring (PDITM). The
present teachings can provide methods for determining the relative
concentration, absolute concentration, or both, of one or more
analytes in one or more samples and provide methods whereby the
relative concentration, absolute concentration, or both, of
multiple analytes in a sample, one or more analytes in multiple
samples, or combinations thereof, can be determined in a multiplex
fashion using mass differential tagging reagents, isobaric tagging
reagents, or both, and mass spectroscopy.
[0037] In embodiments comprising sets of isobaric labels, the
linker group portion can be referred to as a balance group. For
example, a set of four isobaric labels are added to a set of one or
more analytes and combined to form a combined sample that is
subjected to MS/MS analysis to fragment the labeled ketone or
aldehyde compound and produce 4 reporter ions of different mass or
charged analytes. The labels can be made isobaric by an appropriate
combination of heavy atom substitutions of a reporter group or mass
balance group or portion thereof or a mass balance group alone or
portion thereof.
[0038] The heavy atom isotope distribution may generate a different
reporter ion or charged analyte signal in a mass spectrometer. The
ion signals produced by labeled components of a mixture (e.g.,
different analytes, analytes from different samples, standards,
etc.) can be deconvoluted by analyzing the reporter ion signal
associated with the respective label. Deconvolution can determine
the relative and/or absolute amount labeled components in the
mixture. These determinations include time course studies,
biomarker analysis, multiplex analysis, affinity pull-downs, and
multiple control experiments.
[0039] "Parent-daughter ion transition monitoring" or "PDITM" is an
advantageous method of analysis and workflow status, that refers to
a technique whereby the transmitted mass-to-charge (m/z) range of a
first mass separator (often referred to as "MS" or the first
dimension of mass spectrometry) is specifically selected to
transmit a molecular ion (often referred to as "the parent ion" or
"the precursor ion") to an ion fragmentor (e.g. a collision cell,
photodissociation region, etc.) to produce fragment ions (often
referred to as "daughter ions") and the transmitted m/z range of a
second mass separator (often referred to as "MS/MS" or the second
dimension of mass spectrometry) is selected to transmit one or more
daughter ions to a detector which measures the daughter ion signal.
This technique offers unique advantages when the detection of
daughter ions in the spectrum is focused by "parking" the detector
on the expected daughter ion mass. The combination of parent ion
and daughter ion masses monitored can be referred to as the
"parent-daughter ion transition" monitored. The daughter ion signal
at the detector for a given parent ion-daughter ion combination
monitored can be referred to as the "parent-daughter ion transition
signal".
[0040] For example, one embodiment of parent-daughter ion,
transition monitoring is multiple reaction monitoring (MRM) (also
referred to as selective reaction monitoring). In various
embodiments of MRM, the monitoring of a given parent-daughter ion
transition comprises using as the first mass separator (e.g., a
first quadrupole parked on the parent ion m/z of interest) to
transmit the parent ion of interest and using the second mass
separator (e.g., a second quadrupole parked on the daughter ion m/z
of interest) to transmit one or more daughter ions of interest. In
various embodiments, a PDITM can be performed by using the first
mass separator (e.g., a quadrupole parked on a parent ion m/z of
interest) to transmit parent ions and scanning the second mass
separator over a m/z range including the m/z value of the one or
more daughter ions of interest.
[0041] For example, a tandem mass spectrometer (MS/MS) instrument
or, more generally, a multidimensional mass spectrometer (MS.sup.n)
instrument, can be used to perform PDITM, e.g., MRM. Examples of
suitable mass analyzer systems include, but are not limited to,
those that comprise on or more of a triple quadrupole, a
quadrupole-linear ion trap, a quadrupole TOF, and a TOF-TOF.
[0042] In various embodiments, for analyzing one or more ketone or
aldehyde analyte compounds in one or more samples using labels of
the present teachings comprises the steps of: (a) labeling one or
more analyte compounds each with a different label from a set of
labels of formula (II) providing labeled analyte compounds of
formula (III), the labeled analyte compounds each having a mass
balance or reporter ion portion; (b) combining at least a portion
of each of the labeled analyte compounds to produce a combined
sample; (c) subjecting at least a portion of the combined sample to
parent-daughter ion transition monitoring; (d) measuring the ion
signal of one or more of the transmitted analyte or reporter ions;
and (e) determining the concentration of one or more of the labeled
ketone or aldehyde analyte compounds based at least on a comparison
of the measured ion signal of the corresponding analyte or reporter
ion to one or more measured ion signals of a standard compound.
Accordingly, in various embodiments, the concentration of multiple
analyte compounds in one or more samples can be determined in a
multiplex fashion, for example, by combining two or more labeled
analyte compounds to produce a combined sample and subjecting the
combined sample to PDITM, and monitoring the analyte or reporter
ions of two or more of labeled analyte compounds.
[0043] In various embodiments, the step of determining the
concentration of one or more labeled ketone or aldehyde analyte
compounds comprises determining the absolute concentration of one
or more of the labeled ketone or aldehyde analyte compounds,
determining the relative concentration of one or more of the
labeled ketone or analyte compounds, or combinations of both.
[0044] A chromatographic column can be used to separate two or more
labeled analyte compounds. For example, a first labeled analyte
compound found in one or more of the samples is separated by the
chromatographic column from a second labeled analyte compound found
in one or more of the samples. One or more of the samples of
interest can comprise a standard sample containing one or more
standard compounds, wherein the measured ion signal of a reporter
ion corresponding to a standard compound in the method corresponds
to the measured reporter ion signal of one or more labeled standard
compounds in the standard sample.
[0045] In various embodiments of the present teachings, a
concentration curve of a standard compound can be generated by: (a)
providing a non-isotopically enriched standard ketone or aldehyde
compound having a first concentration; (b) labeling the standard
compound with a label from a set of labels wherein the labeled
ketone standard compound has a reporter ion portion; (c) loading at
least a portion of the labeled standard compound on a
chromatographic column; (d) subjecting at least a portion of the
eluent from the chromatographic column to parent-daughter ion
transition monitoring; (e) measuring the ion signal of the
transmitted analyte or reporter ions; (f) repeating steps (a)-(e)
for one or more different standard compound concentrations; and (g)
generating a concentration curve for the standard compound based at
least on the measured ion signal of the transmitted analyte or
reporter ions at two or more standard compound concentrations.
[0046] As will be readily appreciated in the art, the standard
ketone or aldehyde compound can be contained in a standard sample,
and a standard sample can contain more than one standard compound.
As noted above, the sample can be obtained from research, clinical,
agricultural or industrial sources containing a ketone or aldehyde
analyte. In various embodiments, a concentration curve of the
standard compounds can be generated by: (a) providing a standard
sample comprising one or more non-isotopically enriched standard
compounds having first concentrations; (b) adding a label to the
standard sample to label one or more of the standard compounds in
the sample, the labeled standard compounds each having a reporter
ion portion; (c) loading at least a portion of the labeled sample
on a chromatographic column; (d) subjecting at least a portion of
the eluent from the chromatographic column to parent-daughter ion
transition monitoring; (e) measuring the ion signal of the
transmitted analyte or reporter ions; (f) repeating steps (a)-(e)
for one or more different standard samples containing different
concentrations of one or more of the standard compounds; and (g)
generating a concentration curve for one of more of the standard
compounds based at least on the measured ion signal of the
transmitted analyte or reporter ions for the corresponding standard
compound at two or more standard compound concentrations.
[0047] In a preferred embodiment, the step of adding a label to the
standard sample to label one or more of the standard compounds in
the sample comprises a one step reaction where a terminal aminoxy
group forms an oxime with the ketone or aldehyde group of the
analyte standard.
[0048] The phrases "mass differential labels", "mass differential
tags" and "mass differential labeling reagents" are used
interchangeably herein. The phrases "set of mass differential
labels", "set of mass differential tags" are used interchangeably
and refer to, for example, a set of reagents or chemical moieties
where the members of the set (i.e., an individual "mass
differential label" or "mass differential tag") have substantially
similar structural and chemical properties but differ in mass due
to differences in heavy isotope enrichment between members of the
set. Each member of the set of mass differential tags can produce a
different daughter ion signal upon being subjected to ion
fragmentation. Ion fragmentation can be, for example, by collisions
with an inert gas (e.g., collision induced dissociation (CID),
collision a activated dissociation (CAD), etc.), by interaction
with photons resulting in dissociation, (e.g., photoinduced
dissociation (PID)), by collisions with a surface (e.g., surface
induced dissociation (SID)), by interaction with an electron beam
resulting in dissociation (e.g., electron induced dissociation
(EID), electron capture dissociation (ECD)), thermal/black body
infrared radiative dissociation (BIRD), post source decay, or
combinations thereof. A daughter ion of a mass differential tag or
label that can be used to distinguish between members of the set
can be referred to as a reporter ion of the mass differential tag
or label.
[0049] The phrases "isobaric labels", "isobaric tags" and "isobaric
labeling reagents" are used interchangeably. The phrases "set of
isobaric labels", "set of isobaric tags" and "set of isobaric
labeling reagents" are used interchangeably and refer to, for
example, a reagents or chemical moieties where the members of the
set (an individual "isobaric label," "isobaric tag," or "isobaric
labeling reagent") have the identical mass but where each member of
the set can produce a different daughter ion signal upon being
subjected to ion fragmentation (e.g., by collision induced
dissociation (CID), photoinduced dissociation (PID), etc.). A set
of isobaric tags comprises compounds of formula (I) or (II), or a
salt or a hydrate form thereof. A daughter ion of an isobaric tag
that can be used to distinguish between members of the set can be a
reporter ion of the isobaric tag or charged analyte. A set of
isobaric tags is used to label ketone or aldehyde compounds and
produced labeled compounds that are substantially
chromatographically indistinguishable, but which produce signature
ions following CID. The masses of the individual members of a set
of mass labels can be identical or different. Where the individual
isotopic substitutions are the same, the masses can be identical.
Differences in selecting individual atoms for the heavy or light
element incorporated into a specific label of the set can also
yield mass differences based on the specific atomic weights of the
isotopically enriched substituents.
[0050] As used herein, "isotopically enriched" means that a
compound (e.g., labeling reagent) has been enriched synthetically
with one or more heavy atom isotopes (e.g. stable isotopes
including, but not limited to, Deuterium, .sup.13C, .sup.15N,
.sup.18O, .sup.37Cl, or .sup.81Br). Because isotopic enrichment is
not 100% effective, there can be impurities of the compound that
are of lesser states of enrichment and these will have a lower
mass. Likewise, because of over-enrichment (undesired enrichment)
and because of natural isotopic abundance variations, impurities of
greater mass can exist.
[0051] As used herein, "natural isotopic abundance" refers to the
level (or distribution) of one or more isotopes found in a compound
based upon the natural terrestrial prevalence of an isotope or
isotopes in nature. For example, a natural compound obtained from
living plant matter will typically contain about 0.6% .sup.13C.
[0052] The term "substituted" is intended to describe groups having
substituents replacing a hydrogen on one or more atoms, including,
but not limited to, carbon, nitrogen, oxygen, etc., of a molecule.
Substituents can include, for example, alkyl, alkenyl, alkynyl,
halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkoxyl, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic group. Accordingly, the
phrase "a substituent as described herein" or the like refers to
one or more of the above substituents, and combinations
thereof.
[0053] The term "alkyl" includes saturated aliphatic groups, which
includes both "unsubstituted alkyls" and "substituted alkyls", the
latter of which refers to alkyl groups having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone. The term "alkyl" includes straight-chain alkyl groups
(e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl,
nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl,
tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups
(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl),
and cycloalkyl substituted alkyl groups. The term "alkyl" also
includes the side chains of natural and unnatural amino acids.
[0054] An "alkylaryl" or an "aralkyl" group is an alkyl substituted
with an aryl (e.g., phenylmethyl (benzyl)).
[0055] The term "aryl" includes 5- and 6-membered single-ring
aromatic groups, as well as multicyclic aryl groups, e.g.
tricyclic, bicyclic, e.g., naphthalene, anthracene, phenanthrene,
etc.). The aromatic ring(s) can be substituted at one or more ring
positions with such substituents as described above. Aryl groups
can also be fused or bridged with, e.g. alicyclic or heterocyclic
rings which are not aromatic so as to form, e.g. a polycycle.
[0056] The term "alkenyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but which contain at least one double bond. For
example, the term "alkenyl" includes straight-chain alkenyl groups
(e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,
octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,
cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,
cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl
substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl
substituted alkenyl groups. The term alkenyl includes both
"unsubstituted alkenyls" and "substituted alkenyls", the latter of
which refers to alkenyl groups having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone.
[0057] The term "alkynyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but which contain at least one triple bond. For
example, the term "alkynyl" includes straight-chain alkynyl groups
(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl,
octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups,
and cycloalkyl or cycloalkenyl substituted alkynyl groups. The term
alkynyl includes both "unsubstituted alkynyls" and "substituted
alkynyls", the latter of which refers to alkynyl groups having
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone.
[0058] The term "acyl" includes compounds and groups which contain
the acyl (CH.sub.3CO--) or a carbonyl group. The term "substituted
acyl" includes acyl groups having substituents replacing a one or
more of the hydrogen atoms.
[0059] The term "acylamino" includes groups wherein an acyl group
is bonded to an amino group. For example, the term includes
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido
groups.
[0060] The term "aroyl" includes compounds and groups with an aryl
or heteroaromatic group bound to a carbonyl group. Examples of
aroyl groups include phenylcarboxy, naphthyl carboxy, etc.
[0061] The terms "alkoxyalkyl", "alkylaminoalkyl" and
"thioalkoxyalkyl" include alkyl groups, as described above, which
further include oxygen, nitrogen or sulfur atoms replacing one or
more carbons of the hydrocarbon backbone, e.g. oxygen, nitrogen or
sulfur atoms.
[0062] The term "alkoxy" includes substituted and unsubstituted
alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen
atom. Examples of alkoxy groups include methoxy, ethoxy,
isopropyloxy, propoxy, butoxy, and pentoxy groups and may include
cyclic groups such as cyclopentoxy.
[0063] The term "aminoxy" refers to a reactive group on a labeling
reagent having a terminal O--NH.sub.2 group capable of reacting on
a targeted ketone and aldehyde analyte with a ketone or aldehyde
moiety to yield an oxime.
[0064] The term "amine" or "amino" includes compounds where a
nitrogen atom is covalently bonded to at least one carbon or
heteroatom. The term "alkyl amino" includes groups and compounds
wherein the nitrogen is bound to at least one additional alkyl
group. The term "dialkyl amino" includes groups wherein the
nitrogen atom is bound to at least two additional alkyl groups. The
term "arylamino" and "diarylamino" include groups wherein the
nitrogen is bound to at least one or two aryl groups, respectively.
The term "alkylarylamino," "alkylaminoaryl" or "arylaminoalkyl"
refers to an amino group that is bound to at least one alkyl group
and at least one aryl group. The term "alkaminoalkyl" refers to an
alkyl, alkenyl, or alkynyl group bound to a nitrogen atom that is
also bound to an alkyl group.
[0065] The term "amide" or "aminocarboxy" includes compounds or
groups that contain a nitrogen atom that is bound to the carbon of
a carbonyl or a thiocarbonyl group. The term includes
"alkaminocarboxy" groups that include alkyl, alkenyl, or alkynyl
groups bound to an amino group bound to a carboxy group. It
includes arylaminocarboxy groups that include aryl or heteroaryl
groups bound to an amino group which is bound to the carbon of a
carbonyl or thiocarbonyl group. The terms "alkylaminocarboxy,"
"alkenylaminocarboxy," "alkynylaminocarboxy," and
"arylaminocarboxy" include groups wherein alkyl, alkenyl, alkynyl
and aryl groups, respectively, are bound to a nitrogen atom which
is in turn bound to the carbon of a carbonyl group.
[0066] The term "carbonyl" or "carboxy" includes compounds and
groups which contain a carbon connected with a double bond to an
oxygen atom, and tautomeric forms thereof. Examples of groups that
contain a carbonyl include aldehydes, ketones, carboxylic acids,
amides, esters, anhydrides, etc. The term "carboxy group" or
"carbonyl group" refers to groups such as "alkylcarbonyl" groups
wherein an alkyl group is covalently bound to a carbonyl group,
"alkenylcarbonyl" groups wherein an alkenyl group is covalently
bound to a carbonyl group, "alkynylcarbonyl" groups wherein an
alkynyl group is covalently bound to a carbonyl group,
"arylcarbonyl" groups wherein an aryl group is covalently attached
to the carbonyl group. Furthermore, the term also refers to groups
wherein one or more heteroatoms are covalently bonded to the
carbonyl group. For example, the term includes groups such as, for
example, aminocarbonyl groups, (wherein a nitrogen atom is bound to
the carbon of the carbonyl group, e.g., an amide), aminocarbonyloxy
groups, wherein an oxygen and a nitrogen atom are both bond to the
carbon of the carbonyl group (e.g., also referred to as a
"carbamate"). Furthermore, aminocarbonylamino groups (e.g., ureas)
are also include as well as other combinations of carbonyl groups
bound to heteroatoms (e.g., nitrogen, oxygen, sulfur, etc. as well
as carbon atoms). Furthermore, the heteroatom can be further
substituted with one or more alkyl, alkenyl, alkynyl, aryl,
aralkyl, acyl, etc. groups.
[0067] The term "ether" includes compounds or groups that contain
an oxygen bonded to two different carbon atoms or heteroatoms. For
example, the term includes "alkoxyalkyl" which refers to an alkyl,
alkenyl, or alkynyl group covalently bonded to an oxygen atom that
is covalently bonded to another alkyl group.
[0068] The term "ester" includes compounds and groups that contain
a carbon or a heteroatom bound to an oxygen atom that is bonded to
the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl,
alkenyl, or alkynyl groups are as defined above.
[0069] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.-.
[0070] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen, and
oxygen. The term "heterocycle" or "heterocyclic" includes
saturated, unsaturated, aromatic ("heteroaryls" or
"heteroaromatic") and polycyclic rings which contain one or more
heteroatoms. The heterocyclic may be substituted or unsubstituted.
Examples of heterocyclics include, for example, benzodioxazole,
benzofuran, benzoimidazole, benzothiazole, benzothiophene,
benzoxazole, chromene, deazapurine, furan, indole, indolizine,
imidazole, isoxazole, isoindole, isoquinoline, isothiaozole,
methylenedioxyphenyl, napthridine, oxazole, purine, pyran,
pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,
quinoline, tetrazole, thiazole, thiophene, and triazole. Other
heterocycles include morpholino, piprazine, piperidine,
thiomorpholino, and thioazolidine.
[0071] The terms "polycyclic ring" and "polycyclic ring structure"
include groups with two or more rings (e.g., cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which
two or more carbons are common to two adjoining rings, e.g. the
rings are "fused rings". Rings that are joined through non-adjacent
atoms are termed "bridged" rings. Each of the rings of the
polycyclic ring can be substituted with such substituents as
described above.
[0072] As used herein, the term "salt form" includes a salt of a
compound or a mixture of salts of a compound. In addition,
zwitterionic forms of a compound are also included in the term
"salt form." Salts of compounds having an amine, or other basic
group can be obtained, for example, by reaction with a suitable
organic or inorganic acid, such as hydrogen chloride, hydrogen
bromide, acetic acid, perchloric acid and the like. Compounds with
a quaternary ammonium group may also contain a counteranion such as
chloride, bromide, iodide, acetate, perchlorate and the like. Salts
of compounds having a carboxylic acid, or other acidic functional
group, can be prepared by reacting the compound with a suitable
base, for example, a hydroxide base. Accordingly, salts of acidic
functional groups may have a countercation, such as sodium,
potassium, magnesium, calcium, etc.
[0073] As used herein, "hydrate form" refers to any hydration state
of a compound or a mixture or more than one hydration state of a
compound. For example, a labeling reagent discussed herein can be a
hemihydrate, a monohydrate, a dihydrate, etc. Moreover, a sample of
a labeling reagent described herein can comprise monohydrate,
dihydrate and hemihydrate forms.
[0074] Labeling Reagents
[0075] Described herein are sets of mass differential labels of
general formula (I) or (II) as described above. In various
embodiments, provided are sets of isobaric labels of general
formula (I) or (II) in their unsalted and/or unhydrated form. In
various embodiments, the masses of the labels differ by less than
about 0.05 amu in the unsalted and/or unhydrated form. The sets of
labels provided comprise two or more compounds of the general
formula (I) or (II) wherein one or more of the compounds in the set
of labels contains one or more heavy atom isotopes. In various
embodiments, the heavy atom isotopes are each independently
.sup.13C, .sup.15N, .sup.18O, .sup.33S, or .sup.34S.
[0076] The compounds of formula (I) or (II) can be provided in a
wide variety of salt and hydrate forms including, but not limited
to, a mono-TFA salt, a mono HCl salt, a bis-HCl salt, or a bis-TFA
salt, or a hydrate thereof. Variation on formula (I) or (II) are
disclosed in WO2005/068446 which is specifically incorporated by
reference and are generally referred to as iTRAQ reagents.
[0077] In various embodiments, the one or more of the compounds of
the set of labels is isotopically enriched with two or more heavy
atoms; three or more heavy atoms; and/or with four or more heavy
atoms. In various embodiments, a set of labels of formula
incorporated heavy atom isotope such that the isotopes are present
in at least 80 percent isotopic purity, at least 93 percent
isotopic purity, and/or at least 96 percent isotopic purity.
[0078] The reporter group may be comprised of one or more 5, 6 or
7-membered heterocyclic rings as described in U.S. Patent
Application Publication No. US 2008/0014642 A1 which is
specifically incorporated herein in its entirety by reference.
[0079] A set of four isobaric tags of a set of isobaric tags may
comprise a reporter portion and a balance group portion. In various
embodiments, one or more analytes from one or more samples are
labeled with an isobaric tag, the labeled analytes mass filtered
(e.g., with a TOF MS, a RF Multipole MS, a ion mobility MS, etc.)
and subjected to fragmentation (e.g., collision induced
dissociation (CID), photodissociation; etc.) to produce a reporter
ion that can be detected by mass spectrometry.
[0080] Reporter Groups & Ions
[0081] The reporter group portion of an isobaric tag of the present
teachings can be a group that produces a reporter ion from a
labeled analyte when the labeled analyte is subjected to
fragmentation; this reporter ion having a substantially consistent
mass and/or mass-to-charge ratio that can be determined by mass
spectrometry. A charged analyte can also function as a reporter
group for detection by mass analysis. Thus, the reporter group may
be a component of the label reagent or may be the analyte itself.
In some embodiments, the reporter ions of different isobaric
reagent tags in set of isobaric tags have different masses and/or
mass-to-charge ratios (m/z). Different reporter groups, analytes,
standards or ions can comprise one or more heavy atom isotopes to
achieve the differences in mass or m/z between different tags. For
example, heavy atom isotopes of carbon (.sup.12C, .sup.13C, and
.sup.14C), nitrogen (.sup.14N and .sup.15N), oxygen (.sup.16O and
.sup.18O), sulfur (.sup.32S, .sup.33S, and .sup.34S), and/or
hydrogen (hydrogen, deuterium and tritium) can be used in the
preparation of a diverse group of reporter groups and ions.
[0082] Ions of the labeled analyte are fragmented to thereby
produce detectable daughter fragment ions. The detected daughter
ion signal can be used, e.g., to identify the sample from which an
analyte originated. The detected daughter ion signal can be used,
e.g., to determine the relative or absolute amount of analyte in
the sample or samples. The absolute amount is often expressed as a
concentration and/or quantity. For example, the amount of a labeled
analyte in a sample can be determined by comparing the daughter ion
signal to those of other daughter ions, a calibration standard, and
the like. In some embodiments, information such as the amount of
one or more analytes in a particular sample can be associated with
the reporter ion that corresponds to the reporter group of the
isobaric tag used to label each particular sample. The identity of
the analyte or analytes can be correlated with information
pertaining to the different reporter or daughter ions to thereby
facilitate the determination of the identity and amount of each
labeled analyte in one or a plurality of samples.
[0083] When the labeling reagent is comprised of a different
reporter group, the reporter group can comprise a fixed charge or
can be capable of becoming ionized. In various embodiments, use can
be made of a reporter group having a fixed charge or being capable
of being ionized, to isolate and/or use the isobaric tag to label
an analyte in a salt, in a mixture of salts), in zwitterionic form,
or a combination thereof. Ionization of the reporter group
facilitates its determination in a mass spectrometer. When ionized,
the reporter group can comprise one or more net positive or
negative charges. Thus, the reporter group can comprise one or more
acidic groups or basic groups since various embodiments of such
groups can be easily ionized in a mass spectrometer. For example,
the reporter group can comprise one or more basic nitrogen atoms
(positive charge) or one or more ionizable acidic groups such as a
carboxylic acid group, sulfonic acid group or phosphoric acid group
(negative charge). Examples of reporter groups comprising a basic
nitrogen include, but are not limited to, substituted or
unsubstituted, morpholines, piperidines or piperazines.
[0084] Accordingly, the reporter group can be selected to produce a
reporter ion that does not substantially sub-fragment under
conditions typical for the analysis of an analyte. The reporter ion
does not substantially sub-fragment under conditions of
dissociative energy applied to cause fragmentation of the bond
between the nitrogen of the alkyl amide of the reporter group and
the balance group. A reporter that does not "substantially
sub-fragment," means that fragments of the reporter ion are
difficult or impossible to detect above background noise when
applied to the successful analysis of the analyte of interest.
[0085] The mass of a reporter ion can be selected to be different
as compared with the mass of the analyte of interest and/or any of
the expected fragments of the analyte. For example, particularly
where proteins or peptides are the analytes, the reporter ion mass
can be chosen to be different as compared with any naturally
occurring amino acid or peptide, or expected fragments thereof.
[0086] In specific embodiments described herein, the parent ion is
a ketosteroid labeled with an isobaric tag and the daughter ion is
a reporter ion of the isobaric tag. Accordingly, the ion signal of
a reporter ion that is measured at a detector for a given
isobarically labeled steroid parent ion can be referred to as a
"labeled ketosteroid-reporter ion transition signal". Similarly,
the ion signal of a reporter ion that is measured at a detector for
a given isobarically labeled standard compound can be referred to
as a "labeled standard-reporter ion transition signal". Also, where
the label is comprised of a mass balance group, the ion signal of a
charged analyte can be characterized as a "labeled analyte ion
transition signal."
[0087] Balance Groups
[0088] According to various embodiments, isotopes can be used as
balance groups or balance moieties, for example, isotopes of
hydrogen, carbon, nitrogen, oxygen, sulfur, chlorine, bromine, and
the like. Exemplary balance groups or moieties can also include
those described, for example, in U.S. Patent Application
Publications Nos. US 2004/0219685 A1, published Nov. 4, 2004, US
2004/0219686 A1, published Nov. 4, 2004, US 2004/0220412 A1,
published Nov. 4, 2004, and US 2010/0112708 A1, published May 6,
2010, all of which are incorporated herein in their entireties by
reference.
[0089] Applications
[0090] The ketone or aldehyde analyte compounds that can be
analyzed can come from a wide variety of source types such as, for
example, physiological fluid samples, cell or tissue lysate
samples, protein samples, cell culture samples, fermentation broth
media samples, agricultural product samples, animal product
samples, animal feed samples, samples of food or beverage for human
consumption, combinations thereof, and the like. The samples can be
from different sources, conditions, or both; for example, control
vs. experimental, samples from different points in time (e.g. to
form a sequence), disease vs. normal, experimental vs. disease,
contaminated vs. non-contaminated, etc. Examples of physiological
fluids, include, but are not limited to, blood, serum, plasma,
sweat, tears, urine, peritoneal fluid, lymph, vaginal secretion,
semen, spinal fluid, ascetic fluid, saliva, sputum, breast
exudates, and combinations thereof.
[0091] Methods of Labeling
[0092] In various aspects, the present teachings provide methods
for labeling a keto analyte to form a labeled analyte compound. In
various embodiments, the methods comprise reacting a labeling
compound of the general formula (I) or (II) with a
ketone-containing compound. Specifically, ketosteroids were
derivatized with the labeling reagent of formula I and specifically
labeled as in FIG. 1 in 10% acetic acid in MeOH for 30 minutes at
room temperature.
[0093] The present teachings can be applied to both naturally
produced as well as synthetic ketosteroids. Examples of
ketosteroids, including, but not limited to, any steroid,
metabolite or derivation thereof containing a ketone graph, such as
the keto-forms of cortisol, 11-desoxycortisol (compound S),
corticosterone, DHT, testosterone, epitestosterone,
desoxymethyltestosterone (DMT), tetrahydrogestrinone (THG),
estrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone,
16-ketoestradiol, 16 alpha-hydroxyestrone,
2-hydroxyestrone-3-methylether, prednisone, prednisolone,
pregnenolone, progesterone, DHEA (dehydroepiandrosterone), 17 OH
pregnenolone, 17 OH progesterone, 17 OH progesterone, androsterone,
epiandrosterone, and D4A (delta 4 androstenedione).
[0094] Referring to the Examples, FIGS. and Tables below, an
example of labeling ketosteroid analytes such as testosterone,
aldosterene, pregnenolone, and progesterone, with labeling reagents
is shown. In these reactions, the aminoxy moiety reacts with the
ketone or aldehyde on the steroid to form anoxime group on the
labeled compound to yield a labeled analyte.
[0095] Methods of Analysis
[0096] As described herein, methods for determining the
concentration of one or more ketone or aldehyde compounds in two or
more samples are provided by adding a different label to each
sample, combining the differentially labeled samples and using
PDITM to determine a concentration of one or more of the analyte
compounds in the samples. One of the samples may comprise a
standard sample, such as a control sample, a reference sample, a
sample with a compound of known concentration, etc. The methods can
thus provide an analysis of multiple compounds from multiple
samples.
[0097] Certain methods comprise the steps of labeling one or more
ketone or aldehyde compounds, in two or more samples of interest by
adding to each sample of interest a different tag from a set of
tags to form a panel of labeled ketone or aldehyde analyte
compounds. Each tag from the set of tags may comprise a reporter
ion portion as described herein or the ionized analyte may function
as the reporter group. One or more of the labeled ketone or
aldehyde analyte compounds may be differentially labeled with
respect to the sample from which each analyte was obtained or in
which it is contained. The step of adding a label to a ketone or
aldehyde compound may comprise a one step reaction where a first
portion of the label is comprised of the formula Z--R.sub.1,
wherein R.sub.1 is a terminal aminoxy group and Z is a mass
reporter or mass balance group.
[0098] A portion of each of the samples may be combined to produce
a combined sample and a portion thereof analyzed by parent-daughter
ion transition monitoring and measuring the ion signal of one or
more of the transmitted ions. The transmitted parent ion m/z range
includes a m/z value of the labeled analyte compound and the
transmitted daughter ion m/z range includes a m/z value of a
reporter ion derived to the tag of the labeled analyte compound or
is the ionized analyte itself. The concentration of one or more of
the labeled analyte compounds can then be determined based at least
on a comparison of the measured ion signal of the corresponding
transmitter reporter or analyte ions to one or more measured ion
signals of a standard compound. The ion signal(s) can, for example,
be based on the intensity (average, mean, maximum, etc.) of the ion
peak, an area of the ion peak, or a combination thereof. One or
more of the two or more samples of interest can be a standard
sample containing one or more the standard compounds.
[0099] The concentration of a ketone or aldehyde compound is
determined by comparing the measured ion signal of the
corresponding labeled aldehyde ketone analyte compound-reporter ion
transition signal to one or more of:
[0100] (i) a concentration curve for a standard compound-reporter
or analyte ion transition; and
[0101] (ii) a standard compound-reporter ion transition signal for
a standard compound in the combined sample with the labeled ketone
or aldehyde analyte compound.
[0102] PDITM can be performed on a mass analyzer system comprising
a first mass separator, and ion fragmentor and a second mass
separator. The transmitted parent ion m/z range of a PDITM scan
(selected by the first mass separator) is selected to include a m/z
value of one or more of the labeled analyte compounds and the
transmitted daughter ion m/z range of a PDITM scan (selected by the
second mass separator) is selected to include a m/z value one or
more of the reporter ions corresponding to the tag of the
transmitted labeled analyte compound.
[0103] In some embodiments, parent daughter ion transition
monitoring (PDITM) of the labeled analytes is performed using a
triple quadrupole MS platform. More details about PDITM and its use
are described in U.S. Patent Application Publication No. US
2006/0183238 A1, which is incorporated herein in its entirety by
reference. In some embodiments, the aminoxy MS tagging reagent
undergoes neutral loss during MSMS and leaves a reporter ion that
is a charged analyte species. In some embodiments, the aminoxy MS
tagging reagent forms a reporter ion that is a tag fragment during
MSMS.
[0104] The tags added to the two or more samples are selected from
a set of tags within one experimental measurement: (i) multiple
aldehyde or ketone analyte compounds from different samples (e.g.,
a control, treated, time sequence of samples) can be compared
and/or quantified; (ii) multiple concentration measurements can be
determined on the same ketone or aldehyde compound from different
samples; and (iii) different isolates of a clinical sample can be
evaluated against a baseline sample; etc.
[0105] The step of subjecting at least a portion of the combined
sample to PDITM comprises loading the portion of the combined
sample on a chromatographic column (e.g., a LC column, a gas
chromatography (GC) column, or combinations thereof), subjecting at
least a portion of the eluent from the chromatographic column to
parent-daughter ion transition monitoring and measuring the ion
signal of one or more of the transmitted reporter ions.
[0106] The chromatographic column is used to separate two or more
labeled analyte compounds, which differ in the analyte portion of
the labeled compound. For example, a first labeled aldehyde or
ketone compound found in one or more of the samples is separated by
the chromatographic column from a second labeled ketone analyte
compound found in one or more of the samples. Two or more different
labeled analyte compounds are separated such that the different
compounds do not substantially co-elute. Such chromatographic
separation can further facilitate the analysis of multiple
compounds in multiple samples by, for example, providing
chromatographic retention time information on a compound.
[0107] The one or more measured ion signals of a standard compound
used in the step of determining the concentration of one or more of
the labeled analyte compounds can be provided in many ways. In
various embodiments, one or more non-isotopically enriched standard
compounds are labeled with a tag and at least a portion of one or
more of the one or more labeled standard compounds is combined with
at least a portion of each of the labeled analyte compounds to
produce a combined sample; followed by subjecting at least a
portion of this combined sample to PDITM and measuring the ion
signal of one or more of the transmitted reporter ions.
[0108] A tag from the set of tags is added to one or more standard
samples to provide one or more labeled standard samples, each
standard sample containing one or more non-isotopically enriched
standard compounds that are labeled by the tag, the tag added to
the one or more standard samples being different from the tags
added to the samples of interest. At least a portion of one or more
of the one or more labeled standard samples is combined with at
least a portion of each of the samples of interest to produce a
combined sample; followed by subjecting at least a portion of this
combined sample to PDITM and measuring the ion signal of one or
more of the transmitted reporter ions.
[0109] The measured ion signals of one or more of the reporters or
analyte ions corresponding to one or more of the one or more
labeled standard compounds in the combined sample can then be used
in determining the concentration of one or more of the labeled
analyte compounds and can be used to generate a concentration curve
by plotting several values for standard compounds. Accordingly,
determining the concentration of a labeled analyte compound is
based at least on a comparison of the measured ion signal of the
corresponding reporter or analyte ions to the measured ion signal
of one or more reporter or analyte ions corresponding to one or
more of the one or more labeled standard compounds in the combined
sample. The step of subjecting at least a portion of this combined
sample to PDITM can comprise, e.g., a direct introduction into a
mass analyzer system; first loading at least a portion of this
combined sample on a chromatographic column followed by subjecting
at least a portion of the eluent from the chromatographic column to
PDITM and measuring the ion signal of one or more of the
transmitted reporter ions.
[0110] As disclosed herein, PDITM on a standard compound can be
performed on a mass analyzer system comprising a first mass
separator, and ion fragmentor and a second mass separator. The
transmitted parent ion m/z range of a PDITM scan (selected by the
first mass separator) is selected to include a m/z value of one or
more of the labeled standard compounds and the transmitted daughter
ion m/z range of a PDITM scan (selected by the second mass
separator) is selected to include a m/z value one or more of the
reporter or analyte ions corresponding to the transmitted standard
compound.
[0111] Determining the concentration of one or more of the labeled
analyte compounds can be based on both: (i) a comparison of the
measured ion signal of the corresponding reporter or analyte ion to
the measured ion signal of one or more reporter or analyte ions
corresponding to one or more concentration curves of one or more
standard compounds, and (ii) a comparison of the measured ion
signal of the corresponding reporter ion to the measured ion signal
of one or more reporter ions corresponding to one or more labeled
standard compounds combined with the labeled ketone or aldehyde
analyte. A non-isotopically enriched standard compound is provided
having a first concentration and labeled with a tag from the set of
tags is combined with at least a portion of each of the labeled
samples to produce a combined sample, and this combined sample can
then be further analyzed as described herein.
[0112] The present disclosure provides methods for determining the
concentration of one or more ketone or aldehyde analyte compounds
in one or more samples. The methods comprise the steps of labeling
one or more ketone or aldehyde compounds each with a different tag
from a set of tags of formula (I) or (II), wherein R is comprised
of a terminal aminoxy group and Z is comprised of a mass reporter
group or a mass balance group. Where the Z group from each tag from
the set of tags comprises a reporter ion portion, at least a
portion of each of the labeled analyte compound can be combined to
produce a combined sample and at least a portion of the combined
sample can be subjected to parent-daughter ion transition
monitoring (where the transmitted parent ion m/z range includes a
m/z value of the labeled analyte compound and the transmitted
daughter ion m/z range includes a m/z value of a reporter ion
corresponding to the tag of the labeled analyte compound) and
measuring the ion signal of one or more of the transmitted reporter
ions; then determining the concentration of one or more of the
labeled analyte compounds based at least on a comparison of the
measured ion signal of the corresponding reporter ion to one or
more measured ion signals of a standard compound. The ion signal(s)
can, for example, be based on the intensity (average, mean,
maximum, etc.) of the ion peak, an area of the ion peak, or a
combination thereof.
[0113] PDITM can be performed on any suitable mass analyzer known
in the art, including a mass analyzer system comprising a first
mass separator, and ion fragmentor and a second mass separator. The
transmitted parent ion m/z range of a PDITM scan (selected by the
first mass separator) is selected to include a m/z value of one or
more of the labeled analyte compounds and the transmitted daughter
ion m/z range of a PDITM scan (selected by the second mass
separator) is selected to include a m/z value one or more of the
reporter ions corresponding to the tag of the transmitted labeled
analyte compound.
[0114] The one or more ketone or aldehyde compound samples are
labeled with one or more of tags selected from a set of mass
differential tags so that within the same experimental measurement:
(i) multiple ketone or aldehyde containing compounds from different
samples (e.g., a control, treated) can be compared and/or
quantified; (ii) multiple concentration measurements can be
determined on the same ketone or aldehyde compound from the same
sample; and (iii) different isolates of a clinical sample can be
evaluated against a baseline sample.
[0115] The step of subjecting at least a portion of the combined
sample to PDITM comprises introducing the combined sample directly
into a mass analyzer system, e.g., by introduction of the combined
sample in a suitable solution using an electrospray ionization
(ESI) ion source.
[0116] The measured ion signals of one or more of the reporters
ions corresponding to one or more of the one or more labeled
standard compounds in the combined sample determines the
concentration of one or more of the labeled analyte compounds. As
noted above, where the label is comprised of a mass balance group
and the aminoxy group, the charged analyte acts as the reporter
group. Determining the concentration of a labeled analyte compound
is based at least on a comparison of the measured ion signal of the
corresponding reporter or analyte ion to the measured ion signal of
one or more reporter or analyte ions corresponding to one or more
of the one or more labeled standard compounds in the combined
sample. The step of subjecting at least a portion of this combined
sample to PDITM can comprise, e.g., a direct introduction into a
mass analyzer system; first loading at least a portion of this
combined sample on a chromatographic column followed by subjecting
at least a portion of the eluent from the chromatographic column to
PDITM and measuring the ion signal of one or more of the
transmitted reporter or analyte ions; or combinations thereof.
[0117] Determining the concentration of one or more of the labeled
analyte compounds includes a comparison of the measured ion signal
of the corresponding analyte or reporter ion to the measured ion
signal of one or more reporter ions corresponding to one or more
concentration curves of one or more standard compounds. A
non-isotopically enriched standard compound is provided having a
first concentration and labeled with a tag from a set of tags. A
portion of the labeled standard compound is subjected to
parent-daughter ion transition monitoring (where the transmitted
parent ion m/z range includes a m/z value of the labeled standard
compound and the transmitted daughter ion m/z range includes a m/z
value of a reporter or analyte ion corresponding to the tag of the
labeled standard compound) and the ion signal of the reporter or
analyte ion is measured. The steps of labeling and the steps of
PDITM and measuring the ion signal of the transmitted reporter or
analyte ions are repeated for at least one more standard compound
concentration different from the first concentration to generate a
concentration curve for the standard compound.
[0118] The present teachings provide a method for quantifying
ketosteroids and analytes containing keto or aldehyde
functionality. In some embodiments, the method can comprise
derivatization chemistry and a liquid chromatography/tandem mass
spectrometry (LC/MSMS) workflow. The method can comprise using a
permanently charged aminoxy reagent, which significantly increases
the detection limits of ketosteroids. Exemplary aminoxy reagents
that can be used include those of formula (I):
Y--(CH.sub.2)n-ONH.sub.2 (I)
[0119] Wherein Y can be any one of there moieties:
##STR00004##
[0120] In some embodiments, n is an integer from 2 to 50, or from 2
to 20, or from 2 to 10, or from 2 to 6, or from 3 to 8. In some
embodiments, n can be 3 or 4. In some embodiments, Y can be a
different charged moiety than any of these four. Y can be a
permanently charged moiety, for example, a permanently charged
phosphorus-containing or nitrogen-containing moiety. In some
embodiments, Y can be a different charged moiety than those shown
above. In some embodiments, a kit including one or more of the
aminoxy reagents described herein can be provided, for example,
comprising one or more permanently charged aminoxy compounds of
formula (I).
[0121] The method can involve using an MRM workflow for
quantitative analysis of ketosteroids. The reagents can be
isotope-coded for quantitative analysis of an individual or of a
panel of keto compounds. The MS/MS fragmentation at low collision
energies is very clean resulting in one predominant signature ion.
The signature ion can result from a neutral loss from the aminoxy
derivatized product. The MRM transition can be the mass of the
derivatized steroid in Q1 and the mass of the neutral loss fragment
in Q3. The present teachings provide a process for significantly
reducing background noise via derivatization, resulting in improved
sensitivity and targeted selection of Q3 fragments resulting in
improved specificity.
[0122] According to various embodiments, the present teachings
provide a method that reduces or eliminates background noise
without the problems associated with multistep cleanup of a
biological sample and chromatographic separation. In some
embodiments, the method eliminates background noise by utilizing a
derivatization chemistry of ketosteroids with permanently charged
Aminoxy reagents (QAO) and targeted fragmentation that includes
both the reagent and the backbone of the derivatized steroid. The
derivatization with a readily ionized/ionizable molecule results in
better ionization efficiency in ESI MS/MS which increases
sensitivity to the analyte. When the fragment ion that is the Q3
signature ion is selected to include structural fragments with an
attached derivatization reagent, or a part of the reagent, both the
sensitivity and selectivity can be enhanced. The chances that a
compound with exactly the same Q1/Q3 transition would be detected
and create background noise interference are very low. The only
possibility for a similar Q1/Q3 MRM transition would be the
existance of an isobaric ketosteroid in the biological sample. The
isobaric ketosteroid would have to share the same fragmentation
pattern with the analyte in order to appear as interference. In
such a rare scenario, the isobaric ketosteroid can be
chromatographically separated from the analyte.
[0123] According to various embodiments, an added advantage of the
reagent design is that on MSMS fragmentation the reagent generates
a fragment ion, that is, a Q3 signature ion, with a charge on the
derivatized analyte, making it amenable to MS3 analysis. In some
embodiments, the method can be implemented on classes of molecules
with keto- or aldehyde functionality, the detection of which can
benefit from derivatization for ultra high sensitivity analysis by
MS/MS.
[0124] The present teachings provide a highly sensitive and
specific analysis of ketosteroids and classes of molecules
containing a keto functionality. The present teachings provide
higher signal to noise ratios with very low background noise in
MS/MS.
[0125] Kits
[0126] The present invention provides kits for the analysis of
ketone or aldehyde analyte compounds. The kit comprises one or more
labels, including a set of two or more isobaric tags and one or
more reagents, containers, enzymes, buffers and/or instructions for
use. Kits of the present teachings comprise one or more sets of
supports, each support comprising a different isobaric labeling
compound cleavably linked to the support through a cleavable
linker. Examples of cleavable linkages include, but are not limited
to, a chemically or photolytically cleavable linker. The supports
can be reacted with different samples thereby labeling the analytes
of a sample with the isobaric tag associated with the respective
support. Ketone analytes from different samples can be contacted
with different supports and thus labeled with different
reporter/linker combinations.
[0127] According to various embodiments, the kit can comprise a
plurality of different aminoxy tagging reagents, for example, a set
of reagents as described herein. The kit can be configured to
analyze a plurality of different keto or aldehyde analytes, for
example, a plurality of different steroids or ketosteroids, and the
labeling can comprise labeling each with a plurality of different
respective tagging reagents, for example, a different tagging
reagent for each different type of analyte. The analytes to be
analyzed and for which a kit can be configured to detect, can
comprise keto or aldehyde compounds, for example, steroids or
ketosteroids. According to various embodiments of the present
teachings, a kit is provided that comprises one or more aminoxy MS
tagging reagents for tagging one or more ketone or aldehyde
analytes. The aminoxy MS tagging reagent can comprise a compound
having one of the structures described herein.
[0128] The kit can comprise a standard comprising a known ketone or
aldehyde compound, a known steroid, a known ketosteroid, or a
combination thereof. The standard can comprise a known
concentration of a known compound. In some embodiments, the aminoxy
MS tagging reagent included in the kit can comprise one or more
isobaric tags from a set of isobaric tags. In some embodiments, the
kit can comprise a plurality of different isobaric tags from a set
of isobaric tags. In some embodiments, the aminoxy MS tagging
reagent included in the kit can comprise one or more permanently
charged aminoxy reagents from a set of permanently charged aminoxy
reagents. In some embodiments, the kit can comprise a plurality of
different permanently charged aminoxy reagent tags from a set of
permanently charged aminoxy reagent tags.
[0129] The kit can also comprise instructions for labeling the
analyte, for example, paper instructions or instructions formatted
in an electronic file, for example, on a compact disk. The
instructions can be for carrying out an assay. In some embodiments,
the kit can comprise a homogeneous assay in a single container, to
which only a sample need be added. Other components of the kit can
include buffers, other reagents, one or more standards, a mixing
container, one or more liquid chromatography columns, and the
like.
[0130] In some embodiments, a ketosteroid analysis kit is provided
that enables highly sensitive quantitation of ketosteroids from
complex biological matrices, for example, detection in the range of
low pg/mL concentrations.
[0131] Mass Analyzers
[0132] A wide variety of mass analyzer systems can be used in the
present teachings to perform PDITM. Suitable mass analyzer systems
include two mass separators with an ion fragmentor disposed in the
ion flight path between the two mass separators. Examples of
suitable mass separators include, but are not limited to,
quadrupoles, RF multipoles, ion traps, time-of-flight (TOF), and
TOF in conjunction with a timed ion selector. Suitable ion
fragmentors include, but are not limited to, those operating on the
principles of: collision induced dissociation (CID, also referred
to as collisionally assisted dissociation (CAD)), photoinduced
dissociation (PID), surface induced dissociation (SID), post source
decay, by interaction with an electron beam (e.g., electron induced
dissociation (EID), electron capture dissociation (ECD)),
interaction with thermal radiation (e.g., thermal/black body
infrared radiative dissociation (BIRD)), post source decay, or
combinations thereof.
[0133] Examples of suitable mass spectrometry systems for the mass
analyzer include, but are not limited to, those which comprise one
or more of a triple quadrupole, a quadrupole-linear ion trap (e.g.,
4000 Q TRAP.RTM. LC/MS/MS System, Q TRAP.RTM. LC/MS/MS System), a
quadrupole TOF (e.g., QSTAR.RTM. LC/MS/MS System), and a
TOF-TOF.
[0134] In various embodiments, the mass analyzer system comprises a
MALDI ion source. In various embodiments, at least a portion of the
combined sample is mixed with a MALDI matrix material and subjected
to parent-daughter ion transition monitoring using a mass analyzer
with a MALDI ionization source. In various embodiments, at least a
portion of the combined sample loaded on chromatographic column and
at least a portion of the eluent mixed with a MALDI matrix material
and subjected to parent-daughter ion transition monitoring using a
mass analyzer with a MALDI ionization source.
[0135] The mass spectrometer system can comprise a triple
quadrupole mass spectrometer for selecting a parent ion and
detecting fragment daughter ions thereof. In this embodiment, the
first quadrupole selects the parent ion. The second quadrupole is
maintained at a sufficiently high pressure and voltage so that
multiple low energy collisions occur causing some of the parent
ions to fragment. The third quadrupole is selected to transmit the
selected daughter ion to a detector. In various embodiments, a
triple quadrupole mass spectrometer can include an ion trap
disposed between the ion source and the triple quadrupoles. The ion
trap can be set to collect ions (e.g., all ions, ions with specific
m/z ranges, etc.) and after a fill time, transmit the selected ions
to the first quadrupole by pulsing an end electrode to permit the
selected ions to exit the ion trap. Desired fill times can be
determined, e.g., based on the number of ions, charge density
within the ion trap, the time between elution of different
signature peptides, duty cycle, decay rates of excited state
species or multiply charged ions, or combinations thereof.
[0136] One or more of the quadrupoles in a triple quadrupole mass
spectrometer can be configurable as a linear ion trap (e.g., by the
addition of end electrodes to provide a substantially elongate
cylindrical trapping volume within the quadrupole). In various
embodiments, the first quadrupole selects the parent ion. The
second quadrupole is maintained at a sufficiently high collision
gas pressure and voltage so that multiple low energy collisions
occur causing some of the parent ions to fragment. The third
quadrupole is selected to trap fragment ions and, after a fill
time, transmit the selected daughter ion to a detector by pulsing
an end electrode to permit the selected daughter ion to exit the
ion trap. Desired fill times can be determined, e.g., based on the
number of fragment ions, charge density within the ion trap, the
time between elution of different signature peptides, duty cycle,
decay rates of excited state species or multiply charged ions, or
combinations thereof.
[0137] The mass spectrometer system can comprise two quadrupole
mass separators and a TOF mass spectrometer for selecting a parent
ion and detecting fragment daughter ions thereof. In various
embodiments, the first quadrupole selects the parent ion. The
second quadrupole is maintained at a sufficiently high pressure and
voltage so that multiple low energy collisions occur causing some
of the ions to fragment, and the TOF mass spectrometer selects the
daughter ions for detection, e.g., by monitoring the ions across a
mass range which encompasses the daughter ions of interest and
extracted ion chromatograms generated, by deflecting ions that
appear outside of the time window of the selected daughter ions
away from the detector, by time gating the detector to the arrival
time window of the selected daughter ions, or combinations
thereof.
[0138] The mass spectrometer system can comprise two TOF mass
analyzers and an ion fragmentor (such as, for example, CID or SID).
In various embodiments, the first TOF selects the parent ion (e.g.,
by deflecting ions that appear outside the time window of the
selected parent ions away from the fragmentor) for introduction in
the ion fragmentor and the second TOF mass spectrometer selects the
daughter ions for detection, e.g., by monitoring the ions across a
mass range which encompasses the daughter ions of interest and
extracted ion chromatograms generated, by deflecting ions that
appear outside of the time window of the selected daughter ions
away from the detector, by time gating the detector to the arrival
time window of the selected daughter ions, or combinations thereof.
The TOF analyzers can be linear or reflecting analyzers.
[0139] The mass spectrometer system can comprise a tandem MS-MS
instrument comprising a first field-free drift region having a
timed ion selector to select a parent ion of interest, a
fragmentation chamber (or ion fragmentor) to produce daughter ions,
and a mass separator to transmit selected daughter ions for
detection. In various embodiments, the timed ion selector comprises
a pulsed ion deflector. In various embodiments, the ion deflector
can be used as a pulsed ion deflector. The mass separator can
include an ion reflector. In various embodiments, the fragmentation
chamber is a collision cell designed to cause fragmentation of ions
and to delay extraction. In various embodiments, the fragmentation
chamber can also serve as a delayed extraction ion source for the
analysis of the fragment ions by time-of-flight mass
spectrometry.
[0140] In some embodiments, ionization can be used to produce
structurally specific fragment ions and Q3 MRM ions. The labeling
reagent can be wholly or partly contained in the structurally
specific fragment ions. The method can provide both sensitivity and
specificity for the Q3 MRM ions. In some embodiments, ionization
can be sued to produce a dominant neutral loss fragment ion which
can be selected in Q3 and then fragmented to produce structurally
specific ions. These fragment ions can then be used for
identification and quantification in a procedure referred to as
MS3.
EXAMPLES
[0141] Aspects of the present invention may be further understood
in light of the following examples, which are not exhaustive and
which should not be construed as limiting the scope of the present
teachings in any way.
Example 1
Maldi Analysis of Ketosteroids
[0142] A representative synthesis of a labeling reagent, see FIG.
2, is performed as follows:
##STR00005##
[0143] N-(2-N-Boc-hydroxyaminoethyl)phthalimide: To a suspension of
NaH (2.02 g, 50.56 mmol, 60% dispersion in oil) in DMF (25 mL) a
solution of BocNHOH (6.12 g, 133.15 mmol) in DMF (25 mL) was added
dropwise at ambient temperature from an addition funnel under
nitrogen atmosphere. After completion of addition, the reaction
mixture was heated to 55-60.degree. C. for 20 min when a faint
yellow color solution formed. A solution of
N-(2-bromoethyl)phthalimide (7.79 g, 30.64 mmol) in DMF (50 mL) was
then added dropwise to the reaction mixture over 20 min and the
reaction continued at 55-60.degree. C. for another 2 h. After
removal of DMF under reduced pressure, the oil was partitioned
between EtOAc (300 mL) and 0.5 M HCl (150 mL). EtOAc layer was
washed with brine (50 mL), dried over Na.sub.2SO.sub.4, and
concentrated to an oil. The oil was purified by flash
chromatography (40-70% EtOAc in hexanes, 330 g silica column) to
give 1.65 g (18%) of the desired product
(N-(2-N-Boc-hydroxyaminoethyl)phthalimide R.sub.f=0.42 in 30% EtOAc
in Hexanes, silica plate; ES-MS, calculated MH.sup.+=307.1, found
307.1).
##STR00006##
[0144] N-Boc-hydroxyaminoethyl-amine:
N-(2-N-Boc-hydroxyaminoethyl)phthalimide (1.65 g, 5.38 mmol) was
treated with NH.sub.2NH.sub.2 solution in THF (1 M, 25 mL, 25 mmol)
at ambient temperature for 20 h. After removal of solvent and
volatiles under well ventilated condition the solid residue was
treated with 100 mL of dichloromethane, mixed well and filtered.
The solid cake was washed twice with dichloromethane (25 mL).
Combined dichloromethane filtrate was concentrated to a colorless
oil. The oil was purified by flash chromatography (120 g silica
column, 9:1:0.1 dichloromethane-MeOH-Et.sub.3N). Fractions
containing the product (ninhydrin stain) were combined and
concentrate to give a white solid, which was portioned between 6 M
NaOH (50 mL) and dichloromethane (200 mL). Dichloromethane layer
was dried over Na.sub.2SO.sub.4 and concentrated to give the
desired product N-Boc-hydroxyaminoethyl-amine as oil (0.29 g, 31%).
ES-MS, calculated MH.sup.+=177.2, found 177.2.
##STR00007##
Pip-AO-117 and Pip-AO-114: To a solution of iTRAQ-114 and -117
reagents (0.73 mmol) in THF (1 mL) was added a solution of
N-Boc-hydroxyaminoethyl-amine (0.57 mmol) in THF (2 mL) followed by
Et.sub.3N (3.7 mmol), mixed, and heated at 50.degree. C. for 1 h.
TLC(R.sub.f=0.7, 8.5:1:0.1 dichloromethane-MeOH-Et.sub.3N. silica
plates) showed formation of one product which was purified by flash
chromatography (12 g Silica column, 9:1:0.1
dichloromethane-MeOH-Et.sub.3N). Fractions containing pure product
were concentrated and treated with 4 M HCl in dioxane (3 ml) for 45
min. After evaporation of dioxane, the oil was triturated with
dichloromethane to give pip-AO-117 and pip-AO-114 as white solids
(66-70% yields). ES-MS, calculated MH.sup.+=221.2, found 221.2. As
will be readily appreciated by those skilled in the art, synthesis
of reagents 115 and 116 and the non-isotopically enriched species
113 are produced by an analogous synthetic route.
[0145] Using MALDI-MRM instrumentation, highly selective
quantitation of small molecules can be performed at a rate of less
than 5 seconds per sample, eliminating the background noise created
by the MALDI matrix in the low mass range.
[0146] Referring to FIG. 1, the chemical structures and molecular
weights certain ketosteroids are given. To determine the labeling
compound and methods described herein, four representative steroids
were chosen (FIG. 1) Testosterone (Te), Aldosterone (AL),
Pregnenolone (Preg) and Progesterone (Prog). Progesterone was used
as an Internal Standard (IS). As noted above, the derivitization
procedure relies on the animooxy (R) reaction with an aldehyde or
ketone to yield to oxime (X) resulting in a labeled ketone or
aldehyde compound.
[0147] Derivatization Procedure: A mixture of Te, AL and Preg (0.1
mg/mL each) was reacted with the reagent in MeOH+10% AcOH and
incubated for 30 min at RT. The volume of the reaction was 100
.mu.L and the ratio steroid:reagent was 1:500 eqv. Prog was
derivatized separately under similar conditions. The derivatized
steroids were diluted in ACN/H2O before MALDI plate spotting. The
steroid sample (S) is mixed with excess matrix (M) and dried on a
MALDI plate. The plate is loaded onto the sample stage in the ion
source. Laser beam produces matrix neutrals (M), matrix ions
(MH).sup.+, (MH)-, and sample neutrals (S). MALDI plate spotting:
The steroid sample was mixed with the MALDI matrix
.alpha.-cyano-4-hydroxycinnamic acid (CHCA) dissolved in ACN/H2O
1/1 V/V+0.1% TFA (10 mg/mL). 0.75 .mu.L spotted on each well and
air dried. MALDI Instrument and MRM conditions: Analysis was
performed on a 4000 QTRAP.RTM. (ABI; Foster City, Calif.) with
FlashLaser.TM. source which is a high repetition laser optimized
for the analysis of small molecules. The compound dependent and MRM
parameters are described in Table 1. Sample molecules are ionized
by proton transfer from matrix ions: MH++S.fwdarw.M+SH+,
MH-+S.fwdarw.X+SH-. The FlashLaser.TM. source, equipped with a high
repetition laser, generates ultra fast signal from samples spotted
on a target plate.
TABLE-US-00001 TABLE 1 MALDI-MRM Parameters Laser Power, MRM
Steroid Plate Voltage Transition CE (eV) CXP Testosterone 15%, 70V
289.fwdarw.109 35 10 (Te) Derivatized Te 15%, 70V 403.fwdarw.344 30
9 Aldosterone 15%, 70V 361.fwdarw.325 27 16 (AL) Derivatized AL
15%, 70V 475.fwdarw.416 35 9 Pregnenolone 15%, 70V 317.fwdarw.159
47 9 (Preg) Derivatized Preg 15%, 70V 431.fwdarw.372 35 15
Derivatized Prog 15%, 70V 428.fwdarw.369 35 10 (As Internal
Standard)
[0148] FIG. 2 is the chemical structure of reagents designated 114,
115, 116 and 117 having the formula Z--R.sub.1 where R.sub.1 is the
terminal aminoxy O--NH.sub.2 described herein and Z is the mass
reporter group.
[0149] FIGS. 3A-3D are product ion scans of underivatized and
derivatized ketosteroids A: testosterone (CE=30 eV for both); B:
Progesterone (CE=35 and 32 eV respectively); C: Pregnenolone (CE=40
and 35 eV respectively); and D: Aldosterone (CE=35 eV for both). A
comparison of the spectra of derivatized and underivitized ketone
compounds in each of FIGS. 3A, 3B, 3C, and 3D shows the increase in
sensitivity and specificity achieved under high throughput
conditions. Similarly, FIG. 4 shows triplicate MALDI-MRM peaks for
derivatized and underivatized aldosterone, testosterone and
pregnenolone measured for the labeling and analytical methods and
strategies described herein.
[0150] FIG. 5 shows concentration calibration curves for
testosterone, aldosterone and pregnenolone at 0.07-312 pg/spot
generated for mass analysis of the species described above.
TABLE-US-00002 TABLE 2 LOD values and Signal enhancement factor s
of Te, AL and Preg LOD LOD MRM Signal Underivatized Derivatized
Enhancement Ketosteroid pg/spot pg/spot Factor Testosterone 4.8
0.007 700 Aldosterone 12.8 0.04 320 Pregnenolone 45 0.007 6428
[0151] Derivatization of neutral ketosteroids have proven to
enhance significantly their MALDI-MRM sensitivity. Referring to
FIGS. 3A-3D and 4, the improved ionization efficiency resulted in
simplified MS/MS fragmentation as the precursor ion is converted to
only one major product. The combination of instrumental innovation
(high repetition laser for MALDI-MRM) with chemistry (introduction
of an easily ionizable moiety) resulted in a powerful high
throughput, high sensitivity, and high specificity method for
steroids analysis. The ketosteroids investigated in this study
could be detected simultaneously in low fg/spot concentrations
within <5 seconds. Concentration curves resulted in R2>0.99
over >3 orders of magnitude. MALDI spotting and sample clean up
is easily be automated for routine clinical screening and target
analysis.
Example 2
[0152] Derivatization of Ketosteroids and Detection Via LC-MS
[0153] As noted above, the ketone group of a ketosteroid is
derivatized to an oxime functional group (X) by reaction with the
label of the formula Z--R.sub.1. Exclusively protonated molecular
ions are formed without sodium (Na) or potassium (K) additives.
Fragmentation of the derivatized ketosteroid analyte yields a
simplified spectrum with a dominant signature ion at 117 Da
generated from the reporter group. Referring to FIG. 7, the
derivatization procedure described herein takes advantage of the
use of a heavy version as an internal standard for quantitation.
FIG. 7 is a simple flow chart for the quantitative analysis of a
steroid comprising a ketone or aldehyde group as described herein.
Initially, the analyte and, a standard are derivatized by aminoxy
chemistry followed by mixing the labeled analyte and sample. The
mixture is subjected to chromatographic separation, for example, by
LC such as by HPLC, followed by mass analysis by MRM. Quantitation
is enabled by relative or absolute measurement of the signal
derived from one or more analytes and standards. Also, Z may
comprise a mass balance group, such as the quarternary amine shown
in FIG. 6, and the positive charge is transferred to the analyte
which functions as the reporter group to be detected by mass
spectrometry. R.sub.1 remains the terminal aminoxy moiety.
[0154] According to various embodiments, the aminoxy MS tagging
reagents can be used for relative and absolute quantification in
multiplex assays. According to some embodiments, the aminoxy MS
tagging reagents can be used for two-plex, three-plex, four-plex,
and other multi-plex assays. An exemplary method of quantification
is shown with reference to FIG. 7, which illustrates absolute
quantification for a two-plex assay. As described in FIG. 7, the
method can begin with labeling a first sample containing a known
analyte, in this case, a steroid. The first sample can be, for
example, a standard containing a known concentration of a known
steroid. The first sample can be labeled with a first aminoxy tag
from a set of aminoxy tags. Next, a second sample having an unknown
steroid in an unknown concentration can be labeled with a second
aminoxy tag from the same set of aminoxy tags. The labeled first
sample can then be combined with the labeled second sample to form
a mixture.
[0155] Subsequent to mixing, the mixture can be subjected to
separation, such as high performance liquid chromatography (HPLC)
separation, or liquid chromatographic separation on a reversed
phase column. The labeled steroids can elute from the column at
separate times due to their different and distinct retention times
on the column. The peaks eluted from the reversed phase column
comprise peaks that contain the labeled steroids from the first
sample and peaks that contain the labeled steroids from the second
sample. Next, each peak eluted from the column can be subjected to
Parent Daughter Ion Transition Monitoring (PDITM). The ratio of the
signal intensity of peak area of the reporter signals generated
from the first sample, relative to those generated from the second
sample, gives the relative concentration of the steroid in the test
sample. When the concentration of the labeled standard is known,
the specific concentration of the analyte in the sample can be
determined, as shown in FIG. 7.
[0156] A panel of 8 ketosteroids comprised of derivatized and
underivatized testosterone, progesterone, epi-androsterone,
pregnenolone and prednisolone was subjected to LC-MS analysis using
a reverse phase C8 column (Luna C8, 5 .mu.M, phenomenex) and a
water/formic acid gradient over 10 minutes. Mass analysis used an
API 4000 QTRAP in MRM mode. Derivatization was 10% acetic acid in
MeOH at room temperature for 30 minutes.
[0157] Referring to FIGS. 8A-8B, underivatized ketosteroids tend to
distribute the ion current between several species. An example of
cortexolone is shown (FIG. 8A) where strong Na.sup.+ adducts are
observed. Product ion spectra of ketosteroids generated very
complex fragmentation patterns, both the [M+H]+ and [M+Na]+ of
underivatized Cortexolone produced fragment ions distributed across
the entire spectra (FIGS. 8A & 8B).
[0158] Referring to FIG. 8B, in contrast, LC-MS analysis of
derivatized ketosteroids produced strong [M+H]+ with little Na or K
adducts (FIG. 3). Product ion spectra were simplified with a strong
signature from the derivatized analyte at 117 Da. Fragmentation
from the steroid backbone was still present but represented a mere
few percent of the ion current.
[0159] Similarly, FIG. 9 shows an MRM analysis of 1 pg of
testosterone/epi-testosterone in derivatized (a) and underivatized
(b) forms. Sensitivity improvement from the derivatization
procedure is shown for testosterone and epi testosterone. 1 pg of
derivatized testosterone produced a 10.times. increase in
sensitivity compared to underivatized testosterone. A peak shoulder
is observed for derivatized due to separation of the cis/trans
isomer created from the derivatization procedure. The peak at 3.96
mins is dehydro-epi-androsterone which was also presented in the
derivatization mix.
[0160] FIG. 10 shows the reaction efficiency. Reaction efficiency
was evaluated by injecting 300 pg of derivatized testosterone and
epi testosterone and measuring the amount of underivatized analyte.
Reaction efficiency was calculated to be >99%.
[0161] FIG. 11 shows the reaction quantitation. To test
quantitation of the aminoxy reaction, a dilution series (10
fg-10000 fg) and derivatized sample were analyzed. The resultant
calibration curve was linear and compared well to a dilution series
of the underivatized form of the sample (10 fg-10000 fg).
TABLE-US-00003 TABLE 3 Comparison data for derivatized ketosteroids
LOQ LOQ LOQ Derivatized Underivatized Derivatized Analyzed
Ketosteroid (fg) (fg) in PPP Cortexolone 100 10 10 Corticosterone
100 10 <10 17-alpha- 100 1 10 hydroxy-progesterone Progesterone
100 1 10 Pregnenolone 10000 10 10 Prednisolone 200 10 10
Epitestosterone 1000 1 10 Testosterone 100 1 10
[0162] The data shown in the FIGS. and accompanying text show that
the derivatization of ketone compounds using a label of the formula
Z--R.sub.1 using the aminoxy moiety to yield the oxime generated
increases insensitivity by 10-1000 fold depending on the ketone
compound, in this case the representative ketosteroid. The
signature ion at 113 Da can be used as the Q3 mass for MS/MS.
Example 3
[0163] Derivatization of Ketosteroids Using Permanently Charged
Aminoxy Reagent
[0164] FIG. 12 shows the MS/MS fragments and spectrum of QAO
Testosterone using CE=62 eV at which the signature ions contain
fragments from both testosterone structure and from the
derivatizing reagent structure, according to various embodiments of
the present teachings.
[0165] FIG. 13 shows the chromatograms of QAO derivatized
testosterone using an MRM transition of a targeted Q3 fragment as
compared to neutral loss Q3 fragment, according to various
embodiments of the present teachings. Measurement involved using
MRM transitions of neutral loss (403->344) vs. the
reagent-plus-backbone fragment (304->162). As can be seen, lower
detection limits are achievable using a Q3 transition that includes
the reagent and the testosterone backbone, due to a significant
reduction in background noise.
[0166] According to various embodiments of the present teachings,
the method is applied to the targeted fragmentation of a
ketosteroid. For example, FIG. 14 shows the targeted MS/MS
fragmentation and spectrum of QAO Progesterone and the MS/MS
spectrum of QAO Testosterone at CE=62 eV. QAO progesterone
possesses two keto functionalities and therefore results in bis QAO
progesterone.
[0167] FIG. 15 shows the MS/MS spectrum of progesterone at CE=45
eV. FIG. 15 illustrates the background noise reduction in an actual
LC-MS/MS analysis. The MRM transition 272->213 is the neutral
loss from the bis QAO progesterone doubly charged species, and a
high background noise is noticeable. The MRM transition of
272->312.5 is the transition from the doubly charged bis QAO to
a specific fragment that contains part of the reagent structure and
part of the progesterone structure. This MRM transition from a
lower Q1 mass to higher Q3 mass is even more specific and further
improves specificity and reduces background noise in LC-MRM
experiments.
Example 4
Sets of Tagging Reagents
[0168] The following is an exemplary set of four
N-methylpiperazine-aminoxy mass differential reagents, according to
various embodiments of the present teachings:
##STR00008##
[0169] The following is an exemplary set of
N-methylpiperazine-aminoxy isobaric reagents, according to various
embodiments of the present teachings:
##STR00009##
[0170] The following is an exemplary set of quarternary-aminoxy
mass differential reagents, according to various embodiments of the
present teachings:
##STR00010##
[0171] The following is an exemplary set of quarternary-aminoxy
isobaric reagents, according to various embodiments of the present
teachings:
##STR00011##
[0172] All literature and similar material cited in this
application, including, but not limited to, patents, patent
applications, articles, books, treatises, and web pages, regardless
of the format of such literature and similar materials, are
expressly incorporated by reference in their entireties for all
purposes. In the event that one or more of the incorporated
literature and similar materials differs from or contradicts this
application, including but not limited to defined terms, term
usage, described techniques, or the like, this application
controls.
[0173] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described in any way.
[0174] While the present teachings have been described in
conjunction with various embodiments and examples, it is not
intended that the present teachings be limited to such embodiments
or examples. On the contrary, the present teachings encompass
various alternatives, modifications, and equivalents, as will be
appreciated by those of skill in the art.
[0175] The teachings should not be read as limited to the described
order or elements unless stated to that effect. It should be
understood that various changes in form and detail may be made
without departing from the scope of the present teachings. By way
of example, any of the disclosed method steps can be combined with
any of the other disclosed steps to provide a method of analyzing
ring-containing compounds in accordance with various embodiments of
the present teachings. Therefore, all embodiments that come within
the scope and spirit of the present teachings and equivalents
thereto are claimed.
* * * * *