U.S. patent application number 10/444422 was filed with the patent office on 2003-10-23 for protein characterization system.
This patent application is currently assigned to THE MOLECULAR SCIENCES INSTITUTE, INC.. Invention is credited to Brent, Roger, Burbulis, Ian E., Carlson, Robert H..
Application Number | 20030198940 10/444422 |
Document ID | / |
Family ID | 23634237 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030198940 |
Kind Code |
A1 |
Carlson, Robert H. ; et
al. |
October 23, 2003 |
Protein characterization system
Abstract
A system and method for characterizing protein molecules. A
protein molecule of interest is isolated from other types of
protein molecules and modified into a one-dimensional structure.
Each of a first type of amino acid residue of the protein molecule
of interest is labeled with a first tag. Each of a second type of
amino acid residue of the protein molecule is labeled with a second
tag. The first and second tags impart to the protein molecule of
interest a detectable set of characteristic ancillary properties
that function as a fingerprint or characterization of the protein
molecule of interest reflective of the physical structure of the
protein molecule of interest as defined by the amino acid sequence
of the protein molecule of interest. A library listing of the
characterizations corresponding to protein molecules facilitates
identification of protein molecules of interest.
Inventors: |
Carlson, Robert H.; (Union
City, CA) ; Burbulis, Ian E.; (Kensington, CA)
; Brent, Roger; (Berkeley, CA) |
Correspondence
Address: |
Kent S. Burningham, Esq.
TRASKBRITT, PC
Suite 300
230 South 500 East
Salt Lake City
UT
84102
US
|
Assignee: |
THE MOLECULAR SCIENCES INSTITUTE,
INC.
|
Family ID: |
23634237 |
Appl. No.: |
10/444422 |
Filed: |
May 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10444422 |
May 23, 2003 |
|
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|
09412732 |
Oct 5, 1999 |
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6569685 |
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Current U.S.
Class: |
435/4 |
Current CPC
Class: |
G01N 33/542 20130101;
G01N 33/582 20130101; G01N 33/6803 20130101 |
Class at
Publication: |
435/4 |
International
Class: |
C12Q 001/00 |
Claims
What is claimed is:
1. A system for determining a fingerprint of a protein molecule,
the protein molecule including an amino acid residue of a first
type and an amino acid residue of a second type, said system
comprising: (a) denaturation means for linearizing the protein
molecule; (b) labeling means for attaching a tag to the amino acid
residue of the first type in the protein molecule; and (c) detector
means for determining the fingerprint of the protein molecule, the
fingerprint comprising: (i) a first fingerprint constituent
imparted to the protein molecule by said tag when said tag is
attached by said labeling means to the amino acid residue of the
first type in the protein molecule; and (ii) a second fingerprint
constituent imparted to the protein molecule by the amino acid
residue of the second type in the protein molecule.
2. A system as recited in claim 1, wherein a detergent is utilized
by said denaturation means to linearize the protein molecule.
3. A system as recited in claim 2, wherein sodium dodecyl sulfate
is utilized by said denaturation means to linearize the protein
molecule.
4. A system as recited in claim 1, wherein a chaotropic salt is
utilized by said denaturation means to linearize the protein
molecule.
5. A system as recited in claim 1, wherein said tag attached to the
amino acid residue of the first type by said labeling means
comprises a fluorescent dye, said fluorescent dye producing an
emitted radiation when stimulated.
6. A system as recited in claim 5, wherein said detector means
comprises: (a) a source of a primary excitation radiation capable
of stimulating said fluorescent dye to produce said emitted
radiation; and (b) a detector sensitive to said emitted radiation
from said fluorescent dye.
7. A system as recited in claim 6, wherein said detector comprises
a charge coupled device.
8. A system as recited in claim 1, wherein said detector means
comprises: (a) a first source of electromagnetic radiation capable
of stimulating said tag when said tag is attached by said labeling
means to the amino acid residue of the first type in the protein
molecule; and (b) a second source of electromagnetic radiation
capable of stimulating the amino acid residue of the second type in
the protein molecule.
9. A system as recited in claim 8, wherein said detector means
further comprises a charge coupled device so positioned relative to
said first source and said second source as to detect: (a) emitted
radiation from said tag when said tag is attached by said labeling
means to the amino acid residue of the first type in the protein
molecule; and (b) emitted radiation from the amino acid residue of
the second type in the protein molecule.
10. A system as recited in claim 8, wherein said detector means
further comprises a camera so positioned relative to said first
source and said second source as to detect: (a) excitation
radiation from said tag when said tag is attached by said labeling
means to the amino acid residue of the first type in the protein
molecule; and (b) emitted radiation from the amino acid residue of
the second type in the protein molecule.
11. A system as recited in claim 8, wherein said detector means
further comprises a microscope so positioned relative to said first
source and said second source as to detect: (a) excitation
radiation from said tag when said tag is attached by said labeling
means to the amino acid residue of the first type in the protein
molecule; and (b) emitted radiation from the amino acid residue of
the second type in the protein molecule.
12. A system as recited in claim 1, wherein said detector means
comprises: (a) a source of electromagnetic radiation, said
electromagnetic radiation being: (i) capable of stimulating said
tag when said tag is attached by said labeling means to the amino
acid residue of the first type in the protein molecule, and (ii)
capable of stimulating the amino acid residue of the second type in
the protein molecule; and (b) a detector, said detector being: (i)
sensitive to emitted radiation from said first tag when said tag is
attached by said labeling means to the amino acid residue of the
first type in the protein molecule, and (ii) sensitive to emitted
radiation from the amino acid residue of the second type in the
protein molecule.
13. A system as recited in claim 1, wherein said detector means
comprises an atomic force microscope.
14. A system as recited in claim 13, wherein said atomic force
microscope comprises: (a) a detector tip; (b) a donor tag attached
to said detector tip, said donor tag being capable of stimulating
said tag when said tag is attached by said labeling means to the
amino acid residue of the first type in the protein molecule and
said detector tip is in proximity with said tag.
15. A system as recited in claim 1, wherein said detector means
comprises a nuclear magnetic resonance apparatus.
16. A system as recited in claim 15, wherein: (a) a chemical
precursor is utilized by said labeling means to attach said tag to
the amino acid residue of the first type in the protein molecule;
and (b) said tag attached by said labeling means to the amino acid
residue of the first type in the protein molecule is a metal
tag.
17. A system as recited in claim 1, wherein said first type of
amino acid residue in the protein molecule comprises an amino acid
residue selected from a group consisting of cysteine and
lysine.
18. A system as recited in claim 1, wherein the second type of
amino acid residue in the protein molecule comprises
tryptophan.
19. A system for identifying a selected protein molecule in a
sample containing a plurality of protein molecules, the selected
protein molecule including at least one amino acid residue of a
first type and at least one amino acid residue of a second type,
said system comprising: (a) denaturation means for linearizing the
protein molecules in the sample; (b) isolation means for separating
the selected protein molecule from other protein molecules in the
sample; (c) labeling means for attaching a tag to the amino acid
residue of the first type in the protein molecule; (d) detector
means for determining a fingerprint for the selected protein
molecule, said fingerprint comprising: (i) a first fingerprint
constituent imparted to the selected protein molecule by said first
tag when said tag is attached by said labeling means to the amino
acid residue of the first type in the protein molecule; and (ii) a
second fingerprint constituent imparted to the selected protein
molecule by the amino acid residue of the second type in the
protein molecule; and (e) collation means for comparing said
fingerprint to a library of fingerprints for known protein
molecules.
20. A system as recited in claim 19, wherein said isolation means
comprises a hydrodynamic focusing apparatus.
21. A system as recited in claim 19, wherein said isolation means
comprises: (a) a separation plate opaque to light; and (b)
apertures formed through said separation plate.
22. A system as recited in claim 21, wherein each of said apertures
has a diameter of from about 1 nanometer to about 10
nanometers.
23. A system as recited in claim 21, wherein said separation plate
is formed from a material selected from the group consisting of
silicon and opaque plastics.
24. A system as recited in claim 19, wherein said isolation means
comprises an electrophoresis gel.
25. A system as recited in claim 19, wherein said isolation means
comprises a dilute solution.
26. A system as recited in claim 19, wherein said collation means
comprises a computer processor for signals from said detector
means.
27. A system for determining a fingerprint of a protein molecule
having a first type of amino acid residue and a second type of
amino acid residue, said system comprising: (a) a protein molecule
linearization region; (b) a tag attachment region, said tag
attachment region being in fluid association with said
linearization region, in said tag attachment region a tag becoming
attached to each first type of amino acid residue in the protein
molecule; and (c) a detector region in fluid association with and
located downstream of said linearization region and said tag
attachment region, in said detector region constituents of the
fingerprint of the protein molecule being sensed, said constituents
of the fingerprint of the protein molecule comprising: (i) a first
fingerprint constituent of the fingerprint of the protein molecule
imparted to the protein molecule by said tag; and (ii) a second
fingerprint constituent of the fingerprint of the protein molecule
imparted to the protein molecule by the second type of amino acid
residue in the protein molecule.
28. A system as recited in claim 27, wherein said linearization
region is located upstream of said tag attachment region.
29. A protein molecule having an identifiable fingerprint, said
protein molecule comprising: (a) a first amino acid residue, said
first amino acid residue being of a first type; (b) a first tag
attached to said first amino acid residue of said protein molecule,
said first tag imparting to said protein molecule a first
fingerprint constituent; (c) a second amino acid residue, said
second amino acid residue being of a second type; and (d) a second
tag attached to said second amino acid residue of the protein
molecule, said second tag imparting to said protein molecule a
second fingerprint constituent.
30. A protein molecule as recited in claim 29, wherein said protein
molecule further comprises a third amino acid residue of a third
type, said third amino acid residue imparting to said protein
molecule a third fingerprint constituent.
31. A protein molecule as recited in claim 30, wherein said third
type of amino acid residue comprises tryptophan.
32. A protein molecule as recited in claim 29, wherein said protein
molecule comprises a single said first amino acid residue.
33. A protein molecule as recited in claim 29, wherein said protein
molecule comprises: (a) a plurality of said first amino acid
residues; and (b) a plurality of said first tags, individual of
said first tags being attached to corresponding of said first amino
acid residues, said plurality of said first tags collectively
imparting to said protein molecule said first fingerprint
constituent.
34. A protein molecule as recited in claim 30, wherein said protein
molecule comprises a single said third amino acid residue.
35. A protein molecule as recited in claim 30, wherein said protein
molecule comprises a plurality of said third amino acid
residues.
36. A fingerprint for a protein molecule, the protein molecule
including at least one first amino acid residue of a first type, at
least one second amino acid residue of a second type, and at least
one tryptophan amino acid residue, said fingerprint comprising: (a)
a first fingerprint constituent imparted to said protein molecule
by a first tag on each first amino acid residue, said first
fingerprint constituent comprising emitted radiation of a first
wavelength produced by said first tag when said first tag is
stimulated with a first primary excitation radiation; (b) a second
fingerprint constituent imparted to said protein molecule by a
second tag on each second amino acid residue, said second
fingerprint constituent comprising emitted radiation of a second
wavelength produced by said second tag when said second tag is
stimulated with a second primary excitation radiation; (c) a third
fingerprint constituent imparted to said protein molecule by said
first tag on each first amino acid residue, said third fingerprint
constituent comprising a first secondary emitted radiation of said
first wavelength produced by said first tag when said first tag is
stimulated by said emitted radiation of said second wavelength; (d)
a fourth fingerprint constituent imparted to said protein molecule
by each tryptophan amino acid residue, said fourth fingerprint
constituent comprising emitted radiation of a third wavelength
produced by the tryptophan amino acid residue when the said
tryptophan amino acid residue is stimulated with a third primary
excitation radiation; (e) a fifth fingerprint constituent imparted
to said protein molecule by said second tag on each second amino
acid residue, said fifth fingerprint constituent comprising a
secondary emitted radiation of said second wavelength produced by
said second tag when said second tag is stimulated by said emitted
radiation of said third wavelength; and (f) a sixth fingerprint
constituent imparted to said protein molecule by said first tag on
each first amino acid residue, said sixth fingerprint comprising a
second secondary emitted radiation of said first wavelength
produced by said first tag when said first tag is stimulated by
said secondary emitted radiation of said second wavelength.
37. A library of fingerprint values for a set of known protein
molecules, said library comprising: (a) a listing of the identity
of each protein molecule of the set of known protein molecules; (b)
a first fingerprint constituent corresponding to each of said
protein molecules of the set of known protein molecules, said first
fingerprint constituent being representative of the number and
sequence of a first type of amino acid residue in each of said
protein molecules of the set of known protein molecules; and (c) a
second fingerprint constituent corresponding to each of said
protein molecules of the set of known protein molecules, said
second fingerprint constituent representative of the number and
sequence of a second type of amino acid residue in each of said
protein molecules of the set of known protein molecules.
38. A library as recited in claim 37, further comprising a third
fingerprint constituent corresponding to each of said protein
molecules of the set of known protein molecules, said third
fingerprint constituent being representative of the number and
sequence of a third amino acid residue in each of said protein
molecules of the set of known protein molecules.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 412,732 that was filed on Oct. 5, 1999, and
that issued as U.S. Pat. No. 6,569,685 on May 27, 2003.
BACKGROUND
[0002] 1. Field of the Invention
[0003] This invention relates to the rapid identification of
protein molecules by the systematic development for each respective
type of protein molecule of a set of particular, invariant,
readily-detectable distinguishing characteristics, which set of
characteristics will for convenience hereinafter be referred to as
a fingerprint for the corresponding type of protein molecule. The
invention also relates to libraries of different protein molecules
and the corresponding fingerprints therefor, as well as to systems
used in the identification, or fingerprinting, of protein
molecules. The present invention has particular applicability to
the identification of protein molecules obtained from biological
samples.
[0004] 2. Background Art
[0005] There are approximately 100,000 different types of protein
molecules involved in organic processes. Each protein molecule is,
however, comprised of various amino acid building blocks from a
group of about twenty different amino acids. Amino acids chemically
connect end-to-end to form a chain that is referred to as a
peptide. The amino acid building blocks in a peptide chain share as
a group various of the peripheral atomic constituents of each amino
acid. As a result, an amino acid in a peptide chain is not in situ
a complete amino acid. Therefore, an amino acid in a peptide chain
is referred to as an "amino acid residue." A peptide chain becomes
a true protein molecule only when the constituent amino acid
residues have been connected, when certain amino acid residues of
the peptide chain have been modified by the addition to or removal
of certain types of molecules from the functional chemical groups
of these amino acid residues, and when the completed chain of amino
acid residues assumes a particular three-dimensional structure
determined by the sequence of amino acid residues and chemical
modifications thereof.
[0006] Protein molecules do not naturally maintain a
one-dimensional, linear arrangement. The sequence of the amino acid
residues in a protein molecule causes the molecule to assume an
often complex, but characteristic three-dimensional shape. A
protein molecule that has been forced out of this three-dimensional
shape into a one-dimensional, linear arrangement is described as
having been "linearized."
[0007] Protein molecules are involved in virtually every biological
process. Aberrant or mutant forms of protein molecules disrupt
normal biological processes, thereby causing many types of
diseases, including some cancers and inherited disorders, such as
cystic fibrosis and hemophilia. The ability of a protein molecule
to perform its intended function depends, in part, upon the
sequence of amino acid residues of the protein molecule,
modifications to particular amino acid residues of the protein
molecule, and the three-dimensional structure of the protein
molecule.
[0008] Alterations to the sequence of amino acid residues, to the
modifications of particular amino acid residues, or to the
three-dimensional structure of a protein molecule can change the
way in which a protein molecule participates in biological
processes. While many protein molecules and the functions thereof
in biological processes are known, scientists continue the arduous
task of isolating protein molecules, identifying the chemical
composition and structure of each isolated protein molecule, and
determining the functions of the protein molecule, as well as the
consequences of changes in the structures of the protein
molecule.
[0009] The sequence of the amino acid residues in a protein
molecule, which imparts to the protein molecule a unique identity
with a set of unique characteristics, is difficult to detect
rapidly and reliably.
[0010] The identification of a protein molecule typically involves
two steps: (1) purifying the protein molecule; and (2)
characterizing the protein molecule.
[0011] In isolating or purifying protein molecules, a targeted
protein molecule is separated from other, different types of
protein molecules. Some current purification techniques are
sensitive enough to purify an aberrant form of a protein molecule
from normal protein molecules of the same type. Different
purification techniques are based on the different characteristics
of protein molecules, such as the weight of a protein molecule, the
solubility of a protein molecule in water and other solvents, the
reactivity of a protein molecule with various reagents, and the pH
value at which the protein molecule is electrically neutral. The
last is referred to as the isoelectric point of the protein
molecule. Due to the large number of different types of protein
molecules and because some types of protein molecules have very
similar characteristics to other types of protein molecules,
extremely sensitive purification processes are often required to
isolate one type of protein molecule from others. The sensitivity
with which similar types of protein molecules are separated from
each other can be enhanced by combining different types of these
purification techniques.
[0012] In some characterization processes, individual protein
molecules are studied. When characterization processes that permit
one to study individual protein molecules are employed, a single
protein molecule in a sample can be separated or isolated from the
other protein molecules in the sample by diluting the sample.
[0013] Since many purification techniques separate different types
of protein molecules on the bases of the physical or chemical
characteristics of the different types of protein molecules, these
purification techniques may themselves reveal some information
about the identity of a particular type of protein molecule. Once a
particular type of protein molecule has been purified, it may be
necessary to further characterize the purified protein molecule in
order to identify the purified protein molecule. This is
particularly true when attempting to characterize previously
unidentified types of protein molecules, such as aberrant or mutant
forms of a protein molecule.
[0014] Typically, protein molecules are further characterized by
employing techniques that determine the weight of the protein
molecule with increased sensitivity over techniques like gel
electrophoresis, or by determining the sequence of amino acid
residues that make up the protein molecule. One technique that is
useful for performing both of these tasks is mass spectrometry.
[0015] In order to characterize a type of protein molecule by mass
spectrometry, a purified type of protein molecule or a particular
segment of a purified type of protein molecule is given positive
and negative charges, or ionized, and made volatile in a mass
spectrometer. The ionized, volatilized protein molecules or
segments are then analyzed by the mass spectrometer. This produces
a mass spectrum of the protein molecule or segment. The mass
spectrum provides very precise information about the weight of the
protein molecule or segment. Due to the precision with which a mass
spectrometer determines the weight of protein molecules and
segments of protein molecules, when a protein molecule or segment
is analyzed, the information provided by mass spectrometry can be
of use in inferring the sequence of amino acid residues in the
protein molecule or segment. Mass spectrometers are also sensitive
enough to provide information about modifications to particular
amino acid residues of a protein molecule or segment. When a series
of segments from a certain type of protein molecule are analyzed by
mass spectrometry, the information about the sequences of and
modifications to the amino acid residues of each segment can be
combined to infer the sequence of and modifications to amino acid
residues of an entire protein molecule.
[0016] Due to the sensitivity of mass spectrometry and the
resulting ability to infer the sequences of the amino acid residues
and modifications thereto of a particular type of protein molecule,
the differences of aberrant or mutant forms of protein molecules
from a normal protein molecule in amino acid residue sequences and
amino acid residue modifications can also be inferred.
[0017] Nonetheless, mass spectrometry is a time-consuming process
that requires expensive equipment and reagents.
SUMMARY OF THE INVENTION
[0018] It is thus a broad object of the present invention to
increase the speed and efficiency with which protein molecules can
be characterized.
[0019] It is also an object of the present invention to lend to a
protein molecule a characteristic set of ancillary properties that
are rapidly and reliably detectable.
[0020] It is a further object of the present invention to generate
a listing of known protein molecules and their corresponding
fingerprints as provided and determined by the method of the
present invention.
[0021] Achieving the foregoing objects will fulfill further,
broader objects of the present invention of improving biochemical
research and healthcare.
[0022] To achieve the foregoing objects, and in accordance with the
invention as embodied and broadly described herein, systems and
methods for characterizing protein molecules are provided. Also
provided are protein molecules having such tags attached thereto as
impart the protein molecules distinguishing characteristics that
are useable as fingerprints.
[0023] In one form, a system incorporating teachings of the present
invention, which is capable of characterizing a protein molecule,
lends to a protein molecule a characteristic set of ancillary
properties that is rapidly and reliably detectable. As these
ancillary properties are as uniquely identifying of the type of the
protein molecule as fingerprints are reflective of the identity of
a human being, the characteristic set of ancillary properties of a
protein molecule function as a "fingerprint" of the protein
molecule that maybe used to rapidly and reliably identify the type
of the protein molecule.
[0024] A system according to teachings of the present invention has
denaturation means for linearizing the protein molecule, labeling
means for attaching a tag to each of a first type of amino acid
residue of the protein molecule, and detector means for detecting a
fingerprint of the tagged protein molecule. The fingerprint of the
protein molecule has a first fingerprint constituent imparted to
the protein molecule by the tags on each first type of amino acid
residue in the protein molecule and a second fingerprint
constituent imparted to the protein molecule by each second type of
amino acid residue in the protein molecule.
[0025] A system according to teachings of the present invention may
also include isolation means for separating the protein molecule
from other protein molecules in a sample, as well as collation
means for comparing the fingerprint of a protein molecule of
interest to the fingerprints of known protein molecules listed in a
library.
[0026] An example of the denaturation means is a detergent, such as
sodium dodecyl sulfate (hereinafter "SDS"), which gives the entire
protein molecule a negative charge and therefore pulls the protein
molecule out of its three-dimensional structure. Another example of
the denaturation means is .beta.-mercaptoethanol, a chemical that
breaks chemical linkages between the sulfur atoms of two amino acid
residues.
[0027] A protein molecule of interest is separated from the other
types of protein molecules present in a sample by way of isolation
means for separating the protein molecule. Examples of isolation
means that are useful in the systems and methods of the present
invention include, without limitation, hydrodynamic focusing
apparatus, electrophoretic gels, separation plates with apertures
therethrough, and dilution systems for the sample in which the
protein molecule of interest is located.
[0028] In a first example of the labeling means, a fluorescent dye
is attached chemically to the amino acid residues in a protein
molecule of a specific chosen type, thereby forming a tag on each
amino acid residue of the specific chosen type in the protein
molecule. In a second example, the labeling means is a metallic tag
precursor that chemically bonds with the amino acid residues in a
protein of a specific chosen type to form a tag on each amino acid
residue of the specific chosen type in the protein molecule.
[0029] Of the twenty or so types of amino acid residues in protein
molecules, one type of amino acid residue, known as tryptophan,
self-fluoresces when exposed to electromagnetic excitation
radiation of a certain range of wavelengths.
[0030] When a fluorescent dye is used as the labeling means, an
example of the detector means includes electromagnetic excitation
radiation of one or more excitation wavelengths or a range of
excitation wavelengths that will stimulate the tryptophan amino
acid residues of a protein molecule to emit radiation of a first
emitted wavelength. The excitation radiation of the detector means
will also cause the fluorescent dye to emit radiation of a second
emitted wavelength. In this example, the detector means also
includes a detector that is sensitive to the wavelengths of emitted
radiation from the tryptophan amino acid residues of the protein
molecule and to the fluorescent dye.
[0031] When the tags attached to each of the specific type of amino
acid residue of the protein molecule are metallic, the detector
means can include a nuclear magnetic resonance apparatus or other
apparatus known in the art to be capable of detecting single metal
atoms.
[0032] Alternatively, tags can be attached to more than one type of
amino acid residue of the protein molecule. The tags on one type of
amino acid residue are differentially detected from the tags on one
or more other types of amino acid residues to determine different
fingerprint constituents of the protein molecule.
[0033] According to another aspect of the invention, a listing or
database is generated for use with specific protocols to identify
protein molecules. This listing or database is referred to herein
as a library, and includes the identities of a set of known protein
molecules and information about the different fingerprint
constituents of each of the known protein molecules of the listing.
The different fingerprint constituents are imparted to the protein
molecule by the labeling means of the system and detected by way of
the detection means of the system. Collation means for comparing
the fingerprint of a protein molecule of interest to the
fingerprints of the known protein molecules listed in the library
are then used to identify the protein molecule of interest.
Typically, the function of such a collation means can be performed
by a computer processor.
[0034] In yet another aspect, the present invention includes
protein molecules that have been labeled with tags to impart
fingerprint constituents to the protein molecule. Each fingerprint
constituent indicates the number of a particular type of amino acid
residue in a protein molecule and the relative locations of
different types of amino acid residues in the protein molecule.
[0035] The prospect of being able to rapidly and reliably identify
a type of protein molecule has utility in a wide range of research
and clinical applications, such as, for example, in determining
whether or not selected cells of a patient have entered early
stages of cancer.
[0036] Additional objects and advantages of the invention will be
set forth in the description which follows, and in part will be
obvious from the description, or may be learned by the practice of
the invention. The objects and advantages of the invention maybe
realized and obtained by means of the instruments and combinations
particularly pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] A more particular description of the invention briefly
described above will be rendered by reference to a specific
embodiment thereof which is illustrated in the appended drawings in
order to illustrate and describe the manner in which the
above-recited and other advantages and objects of the invention are
obtained. Understanding that these drawings depict only a typical
embodiment of the invention and are not therefore to be considered
limiting of its scope, the invention will be described and
explained with additional specificity and detail through the use of
the accompanying drawings in which:
[0038] FIG. 1 schematically illustrates steps by which a mixed
sample of different types of proteins might routinely be
obtained;
[0039] FIG. 2 is a schematic diagram of a portion of a sequence of
amino acid residues in a typical protein molecule with selected of
those amino acid residues tagged;
[0040] FIG. 3 is a schematic diagram of the portion of the protein
molecule of FIG. 2 with selected of the amino acid residues
therein, including the tagged amino acid residues, symbolized in
simplified form at a higher level of abstraction;
[0041] FIG. 4 is a schematic illustration of the portion of the
protein molecule illustrated in FIG. 3 symbolized with yet enhanced
simplicity at an even higher enhanced level of abstraction;
[0042] FIG. 5 is a schematic illustration of a method incorporating
teachings of the present invention for obtaining a first
fingerprint constituent for a segment of the protein molecule
depicted in FIG. 4 using a single-stage emission produced by
exposing the segment to electromagnetic radiation at a first
excitation wavelength;
[0043] FIGS. 6A and 6B taken together illustrate schematically a
method for obtaining a second fingerprint constituent and a third
fingerprint constituent for the segment of the protein molecule
depicted in FIG. 4 using a two-stage emission produced by exposing
the segment to electromagnetic radiation at a second excitation
wavelength, FIG. 6A illustrating the initial single-stage emission
in the process, and FIG. 6B illustrating the entirety of the
two-stage emission initiated thereby;
[0044] FIGS. 7A-7C taken together illustrate schematically a method
for obtaining fourth, fifth, and sixth fingerprint constituents for
the segment of the protein molecule depicted in FIG. 4 using a
three-stage emission produced by exposing the segment to
electromagnetic radiation at a third excitation wavelength, FIG. 7A
illustrating the initial single-stage emission, FIG. 7B
illustrating the two-stage emission caused thereby, and FIG. 7C
illustrating the entirety of the three-stage emission;
[0045] FIG. 8 is a schematic illustration of a second embodiment of
a method incorporating teachings of the present invention for
obtaining three fingerprint constituents for a segment of the
protein molecule depicted in FIG. 4 using a three-stage emission
produced by exposing the segment to electromagnetic radiation of a
broad range of excitation wavelengths;
[0046] FIG. 9 is a flow chart depicting steps in a method
incorporating teachings of the present invention for determining a
fingerprint for a protein molecule;
[0047] FIG. 10 is a flow chart depicting steps in a method
incorporating teachings of the present invention for identifying a
protein molecule using a fingerprint thereof determined according
to the method of FIG. 9;
[0048] FIG. 11 is a schematic diagram of steps in a first
embodiment of a method for labeling more than one type of amino
acid residue of a protein molecule with tags;
[0049] FIG. 12 is a schematic diagram illustrating steps in a
second embodiment of a method for labeling more than one type of
amino acid residue of a protein molecule with tags and intermediate
structures;
[0050] FIG. 13 is a schematic diagram depicting steps in a third
embodiment of a method for labeling more than one type of amino
acid residue of a protein molecule with tags and intermediate
structures;
[0051] FIG. 14 is a graph depicting fingerprints imparted to two
different types of protein molecules by attaching two different
fluorescent tags to each of two different types of amino acid
residues of the protein molecules;
[0052] FIG. 15 is a schematic representation of a first embodiment
of an apparatus used according to teachings of the present
invention to isolate a protein molecule from other protein
molecules in a sample for the purpose of facilitating the
determination of a fingerprint therefor;
[0053] FIG. 16 is a perspective view of a second embodiment of an
apparatus used according to teachings of the present invention to
isolate a protein molecule from other protein molecules in a sample
for the purpose of facilitating the determination of a fingerprint
therefor;
[0054] FIG. 17 is a perspective view of a third embodiment of an
apparatus used according to teachings of the present invention to
isolate a protein molecule from other protein molecules in a sample
for the purpose of facilitating the determination of a fingerprint
therefor;
[0055] FIG. 18 is an enlarged perspective view of a portion of the
apparatus depicted in FIG. 17;
[0056] FIG. 19 is a schematic representation of an isoelectric
focusing gel used in accordance with teachings of the present
invention to isolate types of protein molecules in a sample of a
plurality of types of protein molecules for the purpose of
identifying the types of protein molecules in the sample;
[0057] FIG. 20 is a schematic representation of an electrophoretic
gel used according to teachings of the present invention to refine
the isolation of types of protein molecules in a sample of a
plurality of types of protein molecules previously separated from
each other with the isoelectric focusing gel depicted in FIG.
19;
[0058] FIG. 21 is a schematic representation plan view of an
electrophoretic gel after the various types of protein molecules in
the sample have been separated from each other into respective
bands in the manner illustrated in FIGS. 19 and 20;
[0059] FIG. 22 is a schematic representation perspective view of a
method for transferring the bands of the electrophoretic gel of
FIG. 21 onto a membrane;
[0060] FIG. 23 is a schematic representation of the membrane of
FIG. 22 after the bands of different types of protein molecules
have been transferred thereto in the manner there illustrated;
[0061] FIG. 24 is a schematic representation of an embodiment of a
system used according to teachings of the present invention for
identifying the types of protein molecules separated into
respective bands in the electrophoretic gel of FIG. 21 and
transferred to the membrane of FIG. 23;
[0062] FIG. 25 is a schematic representation of a first photograph
of the membrane of FIG. 24 obtained by use of the system shown in
FIG. 24 and embodying data for determining a first fingerprint
constituent for each of the types of proteins in the bands in the
membrane of FIG. 24;
[0063] FIG. 26 is a schematic representation of a second photograph
of the membrane shown in FIG. 24 embodying data for determining a
second fingerprint constituent for each of the types of proteins in
the bands in the membrane of FIG. 24;
[0064] FIG. 27 is a schematic representation of a third photograph
of the membrane shown in FIG. 24 embodying data for determining a
third fingerprint constituent for each of the types of proteins in
the bands in the membrane of FIG. 24;
[0065] FIG. 28 is a perspective view of a cover slip for a
microscope slide supporting a drop of a solution containing protein
molecules;
[0066] FIG. 29 is a perspective view of the cover slip depicted in
FIG. 28 inverted and positioned on a microscope slide over a
shallow recess therein, whereby the drop of solution hangs from the
cover slip within the recess;
[0067] FIG. 30 is a perspective view of an apparatus used according
to teachings of the present invention to obtain photographs of a
field of view of a portion of the drop of solution in FIG. 29;
and
[0068] FIGS. 31-33 are schematic representations of a field of view
of a portion of the drop of solution of FIG. 29 obtained by use of
the apparatus of FIG. 30 using different filters, each field of
view embodying data for determining fingerprints for the protein
molecules appearing in that field of view.
DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS
[0069] FIG. 1 depicts steps by which a mixed sample 2 of different
types of protein molecules 4a, 4b, 4c might routinely be obtained
from a living organism 6, such as an animal, a plant, a
microorganism, or the human being depicted. A biological sample 8,
such as tissue or the illustrated cell, is secured from living
organism 6. Mixed sample 2 of protein molecules 4a, 4b, 4c is then
obtained from biological sample 8 by, for example, disrupting the
cell membranes. Protein molecules 4a, 4b, 4c are then linearized
from three-dimensional structures to one-dimensional structures,
such as the linearized protein molecules 10 depicted to the right
in FIG. 1.
[0070] FIG. 2 illustrates a single linearized protein molecule 10.
Protein molecule 10 is a chain of amino acid residues that includes
a number of amino acid residues K of a first type, a number of
amino acid residues C of a second type, a number of tryptophan
amino acid residues W, and a number of amino acid residues X of
other types, many of which are not shown, but only suggested by
ellipsis. A first type of tag 12 is shown chemically attached to
each amino acid residue K of protein molecule 10. A second type of
tag 14 is shown chemically attached to each amino acid residue C.
In FIG. 2 no such tags are attached to tryptophan amino acid
residues W or to other amino acid residues X. Tags 12 and 14 may be
different types of fluorescent tags, different types of metallic
tags, or different types of tags of some other detectable
genre.
[0071] A subcombination of adjacently connected amino acid residues
in protein molecule 10 is identified in FIG. 2 as peptide 11. In
left-to-right sequence, a single amino acid residue W, an amino
acid residue K carrying a tag 12, and an amino acid residue C
carrying a tag 14. As illustrated, amino acid residue W is
tryptophan, amino acid residue K is lysine, and amino acid residue
C is cysteine.
[0072] Protein molecule 10 of FIG. 2 is again depicted in FIG. 3
with selected of the amino acid residues therein, including amino
acid residues W, K, and C, symbolized in simplified form at a
higher level of abstraction. NH.sub.2 represents a first end, or
terminus, of protein molecule 10 and COOH represents a second end
of protein molecule 10.
[0073] FIG. 4 illustrates protein molecule 10 symbolized with yet
enhanced simplicity at an even higher enhanced level of abstraction
relative to that of FIGS. 2 and 3.
[0074] According to one aspect of the teachings of the present
invention, FIGS. 5-7C illustrate a method for characterizing a
protein molecule on the basis of ancillary properties imparted to
the protein molecule by the natural fluorescence of tryptophan
amino acid residues and by fluorescent tags attached to
substantially all of the lysine and cysteine amino acid residues of
the protein molecule.
[0075] For convenience in illustrating the implementation of the
disclosed protein fingerprinting technology, peptide 11 is
illustrated, apart from the balance of protein molecule 10, as a
straight line in FIGS. 5-7C. In the depictions of peptide 11 in
these figures, only the amino acid residue W, K, or C of immediate
concern to the corresponding discussion will be illustrated. As a
further simplification, tag 12 on amino acid residue K and tag 14
on amino acid residue C have been omitted, as was the case in FIGS.
3 and 4. Nonetheless, the depictions in these figures are
illustrative only, and tag 12 and tag 14 should be understood to be
present, respectively, on amino acid residue K and amino acid
residue C.
[0076] For illustrative purposes, tag 12 and tag 14 are tags that
fluoresce when exposed to an appropriate respective wavelength of
electromagnetic radiation. Tryptophan amino acid residue W is
naturally fluorescent, meaning that amino acid residue W will
fluoresce when exposed to an appropriate wavelength of
electromagnetic radiation, even without the attachment thereto of
any fluorescent tag, such as tag 12 or tag 14. Therefore, no such
fluorescent tag is shown on amino acid residues W in FIG. 2 or is
to be suggested in the other of the accompanying figures.
[0077] FIG. 5 illustrates peptide 11 of protein molecule 10 exposed
to a source S of a first primary electromagnetic excitation
radiation 20 of wavelength .lambda..sub.SC. First primary
electromagnetic excitation radiation 20 stimulates the tag on amino
acid residue C to fluoresce, producing emitted radiation 22 of
wavelength .lambda..sub.C.
[0078] The intensity of emitted radiation 22 is measured by a
detector 15 and subjected to a spectral analysis that is reflected
in the graph to the right in FIG. 5. That graph is characterized by
a peak centered about wavelength .lambda..sub.C that serves as a
fingerprint constituent 24 for peptide 11 at wavelength sc.
[0079] Thus, as illustrated in the spectral diagram of FIG. 5, when
a fluorescent tag is chemically attached to amino acid residue C,
first primary electromagnetic excitation radiation 20 of wavelength
.lambda..sub.SC stimulates the emission of a corresponding
fingerprint constituent 24. Nonetheless, the activity reflected in
FIG. 5 is but a depiction of activity related to a single amino
acid residue C in isolation from all other amino acid residues in
peptide 11 or even in protein molecule 10.
[0080] Different approaches are used to obtain a corresponding
fingerprint constituent for the entirety of protein molecule 10 at
wavelength .lambda..sub.SC.
[0081] In a relatively global approach, first primary
electromagnetic excitation radiation 20 is used to illuminate the
entire length of protein molecule 10. The cumulative intensity of
all consequently emitted radiation is measured by a detector and
subjected to an appropriate spectral analysis.
[0082] Alternatively, linearized protein molecule 10 is scrolled
past source S and detector 15. This results in a sequenced series
of fingerprint constituents for protein molecule 10 at wavelength
.lambda..sub.SC. This scrolling process produces markedly greater
information about the structure of protein molecule 10 than does
the global method described previously.
[0083] FIGS. 6A and 6B depict in stages the consequence of the
exposure of peptide 11 of protein molecule 10 to a source S of a
second primary electromagnetic excitation radiation 30 at a
wavelength ?SK that stimulates the tag on amino acid residue K to
fluoresce. The process also stimulates the tag on amino acid
residue C to fluoresce, albeit indirectly. Each of FIGS. 6A and 6B
includes a graph that depicts a corresponding portion of the
response spectra for peptide 11 at wavelength .lambda..sub.SK.
[0084] FIG. 6A illustrates peptide 11 of protein molecule 10
exposed to a source S of a second primary electromagnetic
excitation radiation 30 of wavelength .lambda..sub.SK. Second
Primary excitation radiation 30 causes the tag on amino acid
residue K to fluoresce, producing emitted radiation 32 of
wavelength .lambda..sub.KC.
[0085] The intensity of emitted radiation 32 is measured by
detector 15 and subjected to a spectral analysis that produces the
graph to the right in FIG. 6A. That graph is characterized by a
peak centered about wavelength .lambda..sub.KC. This serves as a
first fingerprint constituent 34 for peptide 11 at wavelength
.lambda..sub.KC.
[0086] Emitted radiation 32 is, however, capable of exciting the
tag on amino acid residue C to fluoresce.
[0087] FIG. 6B illustrates that the exposure of peptide 11 to
emitted radiation 32 excites the tag on amino acid residue C. This
causes the tag on amino acid residue C to fluoresce, producing
emitted radiation 36 of wavelength .lambda..sub.C.
[0088] The intensity of emitted radiation 32 and the intensity of
emitted radiation 36 are measured by detector 15 and subjected to a
spectral analysis that produces the graph to the right in FIG. 6B.
That graph is characterized not only by first fingerprint
constituent 34, but by a second peak centered about wavelength
.lambda..sub.C. The latter serves as a second fingerprint
constituent 38 for peptide 11 at wavelength .lambda..sub.SK.
[0089] For emitted radiation 32 to have the illustrated effect on
the tag on amino acid residue C, the tag on amino acid residue K
that produced emitted radiation 32 must be located relatively
proximately along protein molecule 10 to the tag on amino acid
residue C.
[0090] Thus, as illustrated in the spectral diagram of FIG. 6B,
when fluorescent tags are chemically attached to two different
types of amino acid residues K and C, second primary
electromagnetic excitation radiation 30 of wavelength
.lambda..sub.SK that excites the tag on amino acid residue K will
stimulate the emission of two corresponding additional fingerprint
constituents for peptide 11.
[0091] FIGS. 7A-7C illustrate in stages the consequence of the
exposure of peptide 11 of protein molecule 10 to a source S of a
third primary electromagnetic excitation radiation 40 at a
wavelength .lambda..sub.SW that stimulates tryptophan amino acid
residue W to fluoresce. The process also indirectly stimulates the
tags on amino acid residues K and C of peptide 11 to fluoresce.
Each of FIGS. 7A-7C includes a graph that depicts a corresponding
portion of the response spectra for peptide 11 at wavelength
.lambda..sub.SW.
[0092] In FIG. 7A it can be seen that the exposure of peptide 11 to
third primary electromagnetic excitation radiation 40 excites
tryptophan amino acid residue W. This causes tryptophan amino acid
residue W to fluoresce, producing emitted radiation 42 of
wavelength .lambda..sub.WK. The intensity of emitted radiation 42
is measured by detector 15 and subjected to a spectral analysis
that produces the graph to the right in FIG. 7A. That graph is
characterized by a peak centered about wavelength .lambda..sub.WK
that serves as a first fingerprint constituent 44 for peptide 11 at
wavelength .lambda..sub.SW.
[0093] Emitted radiation 42 of FIG. 7A is, however, radiation that
is capable of exciting the tag on amino acid residue K to
fluoresce.
[0094] In FIG. 7B it can be seen that the exposure of peptide 11 to
emitted radiation 42 excites the tag on amino acid residue K. This
causes the tag on amino acid residue K to fluoresce, producing
emitted radiation 46 of wavelength .lambda..sub.KC. The intensity
of emitted radiation 46 is measured by detector 15 and subjected to
a spectral analysis that produces the graph to the right in FIG.
7B. That graph is characterized, not only by first fingerprint
constituent 44, but by a second peak centered about wavelength
.lambda..sub.KC. The latter serves as a second fingerprint
constituent 48 for peptide 11 at wavelength .lambda..sub.SW.
[0095] For emitted radiation 42 to have the illustrated effect on
the tag on amino acid residue K, tryptophan amino acid residue W
that produced emitted radiation 42 must be located relatively
proximately along protein molecule 10 to the tag on amino acid
residue K.
[0096] Emitted radiation 46 of FIG. 7B is, however, radiation that
is capable of exciting the tag on amino acid residue C to
fluoresce.
[0097] In FIG. 7C it can be seen that the exposure of peptide 11 to
emitted radiation 46 excites the tag on amino acid residue C. This
causes the tag on amino acid residue C to fluoresce, producing
emitted radiation 50 of wavelength .lambda..sub.C. The intensity of
emitted radiation 50 is measured by detector 15 and subjected to a
spectral analysis that produces the graph to the right in FIG. 7C.
That graph is characterized not only by first fingerprint
constituent 44 and second fingerprint constituent 48, but by a
third peak centered about wavelength .lambda..sub.C. The latter
serves as a third fingerprint constituent 52 for peptide 11 at
wavelength .lambda..sub.SW.
[0098] For emitted radiation 46 to have the illustrated effect on
the tag on amino acid residue C, the tag on amino acid residue K
that produced emitted radiation 46 must be located relatively
proximately along protein molecule 10 to the tag on amino acid
residue C.
[0099] Thus, as illustrated in the spectral diagram of FIG. 7C,
when fluorescent tags are chemically attached on two different
types of amino acid residues K and C, third primary electromagnetic
excitation radiation 40 of wavelength .lambda..sub.SW that excites
tryptophan amino acid residue W will stimulate the emission of
three corresponding fingerprint constituents for peptide 11 at
wavelength .lambda..sub.SW.
[0100] Fingerprint constituents 24, 34, 38, 44, 48, and 52 together
comprise one possible fingerprint for peptide 11, or by comparison
for protein molecule 10. Fingerprint constituents are obtained for
additional known protein molecules and collected in a computer
database. The database then serves as a library of fingerprints for
a set of protein molecules with tags on amino acid residues K, C
when exposed to primary excitation radiations of wavelengths
.lambda..sub.SC, .lambda..sub.SK, and .lambda..sub.SW.
[0101] An unknown protein molecule is identified by chemically
attaching the same types of tags to all corresponding types of
amino acid residues of that protein molecule. Fingerprint
constituents of the unknown protein molecule are determined using
the methodology described. The fingerprint constituents of the
unknown protein molecule are compared to the entries in the library
of protein fingerprints. If a matching set of protein fingerprints
is located in the library, the unknown protein molecule is
identified.
[0102] The disclosed fingerprinting method can be used to rapidly
identify a plurality of unknown protein molecules in a mixture of
protein molecules, such as those contained within a sample
cell.
[0103] In a variation of the fingerprinting method illustrated in
FIGS. 4-7C, shown in FIG. 8, a single source S that emits a broad
spectrum of wavelengths of primary electromagnetic excitation
radiation 60, including wavelengths .lambda..sub.SW,
.lambda..sub.SK, and .lambda..sub.SC, is used to simultaneously
stimulate amino acid residues W, K, and C of peptide 11 to
fluoresce.
[0104] The process also indirectly stimulates the tags on amino
acid residues K and C of peptide 11 to fluoresce. FIG. 8 includes a
graph that depicts a corresponding portion of the response spectra
for peptide 11 at wavelengths .lambda..sub.SW, .lambda..sub.SK, and
.lambda..sub.SX.
[0105] In FIG. 8 it can be seen that the exposure of peptide 1 to
primary electromagnetic excitation radiation 60 excites tryptophan
amino acid residue W. This causes tryptophan amino acid residue W
to fluoresce, producing emitted radiation 62 of wavelength
.lambda..sub.WK.
[0106] Emitted radiation 62 is radiation that is capable of
exciting the tag on amino acid residue K to fluoresce. In addition,
wavelength .lambda..sub.SK of primary electromagnetic excitation
radiation 60 from source S causes the tag on amino acid residue K
to fluoresce. When stimulated either by primary electromagnetic
excitation radiation 60 or by emitted radiation 62, the tag on
amino acid residue K produces emitted radiation 66 of wavelength
.lambda..sub.KC.
[0107] Emitted radiation 66 is capable of exciting the tag on amino
acid residue C to fluoresce. In addition, wavelength
.lambda..sub.SC of primary electromagnetic excitation radiation 60
from source S causes the tag on amino acid residue C to fluoresce.
When stimulated either by primary electromagnetic excitation
radiation 60 or by emitted radiation 66, the tag on amino acid
residue C produces emitted radiation 70 of wavelength
.lambda..sub.C.
[0108] The intensities of emitted radiation 62, 66, 70 are measured
by detector 15 and subjected to a spectral analysis that produces
the graph to the right in FIG. 8. That graph is characterized by a
first peak centered about wavelength
.lambda..sub.WK/.lambda..sub.SK that serves as a first fingerprint
constituent 64 for peptide 11, by a second peak centered about
wavelength .lambda..sub.KC/.lambda..sub.KC that serves as a second
fingerprint constituent 68 for peptide 11, and by a third peak
centered about wavelength .lambda..sub.C that serves as a first
fingerprint constituent 72 for peptide 11 when peptide 11 is
exposed to primary electromagnetic excitation radiation 60 that
includes wavelengths .lambda..sub.SW, .lambda..sub.SK, and
.lambda..sub.SC.
[0109] In another aspect of the present invention, the
characterization of a protein molecule in a manner that
incorporates teachings of the present invention is but a step in a
method in which proteins are isolated and characterized. FIG. 9 is
a flow chart that illustrates, by way of example and not by way of
limitation, one such method. The characterization steps of FIG. 9
are broader than the characterization steps illustrated in FIGS.
5-8. Each of the boxes of the flow chart of FIG. 9 represents a
general step of the method. The order of the boxes is not intended
to be limiting, neither is any single step illustrated, as some of
the steps are optional.
[0110] In box 74, the protein molecules in a sample are exposed to
one or more chemicals to convert the protein molecules from native
three-dimensional structures to linear, one-dimensional structures.
These chemicals are referred to herein as denaturation means for
linearizing the protein molecule. When the structure of a protein
molecule has been modified in this manner, the protein molecule is
said to have been "linearized."
[0111] By way of example and not limitation, linearization means
according to the invention can include the use of chemicals that
are known to be useful in linearizing a protein molecule. These
could include, without limiting the scope of the invention, ionic
detergents, such as SDS, and nondetergents, such as the chaotropic
salts guanadinium and urea. The chemical known as
.beta.-mercaptoethanol, which breaks chemical bonds that can form
between sulfur atoms of two amino acid residues, such as methionine
and cysteine amino acid residues, can also be used as linearization
means.
[0112] Alternatively, it may be desirable to analyze a protein
molecule in the native three-dimensional structure thereof or
without disrupting chemical bonds between sulfur atoms of two amino
acid residues.
[0113] In box 76, each of a first type of amino acid residue of the
protein molecule is labeled with a first tag. The amino acid
residues are labeled with tags, such as fluorescent tags or
metallic tags. Specific tags can be attached to specific amino acid
residues by way of known chemical reactions. Alternatively, the
first type of amino acid residues of the protein molecule can be
labeled prior to the linearization step depicted in box 74.
[0114] In box 78, the protein molecule or type of protein molecule
to be examined is isolated from the other protein molecules or
types of protein molecules in the sample. The isolation step
depicted in box 78 can occur before or after the linearization step
depicted in box 74, or before or after the labeling step depicted
in box 76.
[0115] Once the protein molecule or type of protein molecule to be
examined is isolated, the protein molecule can be characterized. In
box 80, a second type of amino acid residue of the protein molecule
is detected. The second type of amino acid residue can itself be
detected, or some signal generated by the second type of amino acid
residue can be detected. When fluorescent dyes are employed as the
tags on amino acid residues of the first type, the radiation
generated due to the self-fluorescence of amino acid residue W when
excited by radiation, such as radiation of wavelength
.lambda..sub.SW, is detected, using, for example, the methods
illustrated in FIGS. 7A and 8.
[0116] Next, in box 82, an interaction between the second type of
amino acid residue and the first tag on the first type of amino
acid residue is detected. For example, when the first tag is a
fluorescent tag as illustrated in FIGS. 7B and 8, the second type
of amino acid residue, in this example amino acid residue W, emits
radiation of a wavelength .lambda..sub.WK, which can excite the
first tag on the first type of amino acid residue, in this example
amino acid residue K.
[0117] The first tag on the first type of amino acid residue of the
protein molecule is then detected, as depicted by box 84 of the
flow chart of FIG. 9. When the first tag is a fluorescent tag, the
first tag can be detected by the methods illustrated in FIGS. 6A
and 8, wherein peptide 11 is exposed to excitation radiation having
a wavelength .lambda..sub.SK that will stimulate the first tag to
emit detectable radiation.
[0118] In box 86, the data obtained from each of the steps depicted
by boxes 80, 82, and 84 is recorded. Data can be recorded in any
manner known in the art, such as manually or in a computer
database.
[0119] According to another aspect, the present invention includes
a method for identifying an unknown protein molecule. FIG. 10 is a
flow chart that depicts exemplary steps that may be carried out to
perform the method. Each of the boxes of the flow chart of FIG. 10
represents a general step of the method. The order in which the
boxes are presented in FIG. 10 is not meant to be limiting.
Moreover, not all of the steps are required in performing the
method.
[0120] In box 90, a mixture of protein molecules is obtained from a
sample. Referring again to FIG. 1, biological sample 8 can be
obtained from living organism 6 by known processes. Protein
molecules 4a, 4b, 4c can then be removed from biological sample 8
by extraction processes that are known to those in the art.
[0121] In box 92 of FIG. 10, the protein molecules obtained from a
biological sample are linearized in the same manner as described in
reference to box 74 of FIG. 9. The linearization step of box 92 is
optional, as it may be desirable to leave the protein molecule
completely or partially in the natural three-dimensional
configuration thereof.
[0122] One or more of the types of amino acid residues of the
protein molecule are labeled at box 94. The amino acid residues are
labeled with known tags, such as fluorescent tags or metallic tags.
Specific tags can be attached to specific amino acid residues by
way of known chemical reactions. Accordingly, different fluorescent
dyes can be attached to different types of amino acid residues.
Alternatively or in addition, one or more of the types of amino
acid residues of a protein molecule can be labeled with metallic
tags.
[0123] As an example, FIG. 2 illustrates each amino acid residue K
as having attached thereto a first tag 12. Each amino acid residue
C shown in FIG. 2 has a second tag 14 thereon. When different tags
are used on different types of amino acid residues, the numbers and
relative locations of each of the different types of amino acid
residues can be distinguished from each other. FIGS. 5-8 illustrate
an example of how different tags on different types of amino acid
residues are used to characterize a protein molecule.
[0124] Different types of amino acid residues in protein molecules
are labeled using various methods. A first embodiment of such a
method is shown in FIG. 11 by way of illustration and not
limitation. A cysteine reactive fluorescent tag 14 is attached to
each amino acid residue C of peptide 11 by known processes. A
different, lysine reactive fluorescent tag 12 is attached to each
amino acid residue K of peptide 11, also by known processes.
[0125] FIG. 12 depicts, by way of illustration and not limitation,
a second embodiment of labeling method conducted according to the
teachings of the present invention. In the second embodiment, a
cysteine reactive amino group 110 having the chemical formula
--CH.sub.2NH.sub.2 is attached to each amino acid residue C of
peptide 11. Cysteine reactive amino group 110 permits lysine
reactive groups to be attached to amino acid residue C. A lysine
reactive fluorescent tag 12 is then attached to each amino acid
residue K and to cysteine reactive amino group 110 on each amino
acid residue C of peptide 11. The emitted radiation from tags 12 is
detected to provide a first fingerprint constituent of peptide 11.
After the emitted radiation of tag 12 is detected, tags 12 attached
to cysteine reactive amino groups 110 on amino acid residues C can
be removed by use of a hydrolyzing reagent 112, as known in the
art. Tags 12 remaining only on amino acid residues K can then be
detected to provide a second fingerprint constituent of peptide
11.
[0126] A third embodiment of labeling method according to teachings
of the present invention is shown in FIG. 13 by way of illustration
and not limitation. First, a cysteine reactive blocking group 114
of a type known in the art is chemically attached to each amino
acid residue C of peptide 11. A lysine reactive fluorescent tag 12
is then attached to each amino acid residue K of peptide 11.
Blocking group 114 is then removed from each amino acid residue C
by use of a hydrolyzing reagent 112, as known in the art. Next, a
different fluorescent tag 116 that can react with either amino acid
residue K or amino acid residue C is then chemically attached to
each amino acid residue C.
[0127] Returning to the inventive method illustrated in FIG. 10,
box 96 depicts the isolation of a protein molecule from other
protein molecules in the sample, or of a type of protein molecule
from other types of protein molecules in the sample. The isolation
of a protein molecule from other protein molecules or of a type of
protein molecule from other types of protein molecules can occur
before or after the linearization step depicted in box 92, or
before or after the labeling step depicted in box 94.
[0128] Once the desired protein molecule or type of protein
molecule has been isolated, the protein molecule or type of protein
molecule is characterized at box 98. In characterizing a protein
molecule or type of protein molecule, a fingerprint of the protein
molecule is determined. FIGS. 5-7B and 8 illustrate examples of a
method for determining the fingerprint of a protein molecule, in
which amino acid residues of the protein molecule are labeled with
fluorescent tags.
[0129] When the fingerprint of a protein molecule of interest has
been determined, that fingerprint is compared at box 100 to the
fingerprints in a library of fingerprints. The library of
fingerprints has a listing of known protein molecules. Each of the
known protein molecules in the listing has a corresponding
fingerprint that was determined by the same processes used to
determine the fingerprint of the protein molecule of interest.
Thus, when the fingerprint of the protein molecule of interest
matches with a fingerprint in the library, the protein molecule of
interest is identified.
[0130] FIG. 14 is a graph that illustrates the fluorescence
spectra, or fingerprints 120, 122, of two different protein
molecules. Fingerprints 120, 122 were obtained by the method
illustrated in FIG. 8. Fingerprint 120 is characteristic of the
protein molecule known as bovine serum albumin (hereinafter "BSA"),
while fingerprint 122 corresponds to the protein molecule known as
alcohol dehydrogenase (hereinafter "ADH"). Each of fingerprints
120, 122 includes a pair of major intensity peaks, respectively at
the same wavelengths.
[0131] Fingerprints 120, 122 are imparted to BSA and to ADH by way
of the self-fluorescence of each of the tryptophan amino acid
residues, the fluorescence of napthalenedicarboxyaldehyde
(hereinafter "NDA") tags on each of the lysine amino acid residues,
and the fluorescence of rhodamine tags on each of the cysteine
amino acid residues of BSA and of ADH.
[0132] The present invention includes various approaches to
isolating a protein molecule of interest and to determining a
fingerprint of the protein molecule of interest. Structures capable
of performing each of these functions are respectively referred to
as isolation means for separating the protein molecule and detector
means for detecting the fingerprint of the protein molecule. FIGS.
15-33 illustrate, by way of example and not limitation, various
combinations of structures for performing the functions of an
isolation means and a detector means according to teachings of the
present invention, thereby to isolate and determine a fingerprint
of a protein molecule of interest.
[0133] FIG. 15 illustrates a first embodiment of detector means
that can be used according to teachings of the present invention to
isolate a protein molecule from other protein molecules in a sample
for the purpose of characterizing or identifying the protein
molecule. The apparatus depicted in FIG. 15 is a hydrodynamic
focusing apparatus 130 that isolates individual protein molecules
from each other.
[0134] Hydrodynamic focusing apparatus 130 has a first region 132
into which sample 2 of one or more protein molecules is introduced.
From first region 132, sample 2 flows into a second region 134 of
hydrodynamic focusing apparatus 130, where the protein molecules in
sample 2 are linearized and labeled with tags by way of chemicals
introduced into second region 134 by way of inlets 135. Next,
sample 2 flows into a third region 136, where the different types
of protein molecules in sample 2 are separated from each other.
Third region 136 can have therein a small separation column, a
microelectrophoresis gel, or other apparatus known to be capable of
separating different types of protein molecules. An eluent 137 that
includes the different types of protein molecules 4a, 4b, 4c from
sample 2 flows from the separation apparatus of third region 136
into a fourth region 138 of hydrodynamic focusing apparatus 130.
Different types of protein molecules 4a, 4b, 4c elute separately
from the separation apparatus of third region 136 into fourth
region 138.
[0135] Eluent 137, which contains separated types of protein
molecules 4a, 4b, 4c, flows through fourth region 138, into a
laminar flow region 140 of hydrodynamic focusing apparatus 130. Two
opposing inlets 142 communicate with laminar flow region 140 to
permit the introduction of a buffer 144 into laminar flow region
140. Buffer 144 is introduced under pressure to create a laminar
flow of buffer 144 and eluent 137 as the flow paths of eluent 137
and buffer 144 merge. Due to the laminar flow of buffer 144 into
eluent 137, eluent 137 flows in a thin layer 146 between two layers
148 of buffer 144. When buffer 144 is introduced into laminar flow
region 140 under sufficient pressure, individual protein molecules
4a, 4b, 4c are isolated by the laminar flow of buffer 144 into
eluent 137.
[0136] As protein molecules 4a, 4b, 4c flow in thin layer 146
through a detection region 149 of hydrodynamic focusing apparatus
130, a fingerprint can be determined that is imparted to each of
protein molecules 4a, 4b, 4c in accordance with teachings of the
present invention.
[0137] FIG. 16 depicts a second embodiment of an apparatus used
according to teachings of the present invention to isolate and
characterize a protein molecule. The apparatus of FIG. 16 is an
atomic force microscope 150. Individual protein molecules 10 are
separately analyzed with atomic force microscope 150 due to the
atomic resolution of atomic force microscope 150.
[0138] Protein molecules 10 are diluted and placed on a support 152
with distinct protein molecules 10 separated from one another.
Support 152 is formed from a material, such as mica or glass, to
which protein molecules 10 will adhere and upon which protein
molecules 10 will be immobilized.
[0139] Atomic force microscope 150 has a cantilever 154 with a
detector tip 156. As detector tip 156 is brought into proximity
with a selected protein molecule 10, interactions between detector
tip 156 and chemical structures of selected protein molecule 10
cause cantilever 154 to vibrate. The vibrations of cantilever 154
are measured by way of a laser detection system 158 that directs a
laser beam L onto cantilever 154 and detects vibrations of laser
beam L as the same is reflected by cantilever 154. The measurements
are used to determine the molecular weight and length of selected
protein molecule 10.
[0140] Detector tip 156 has attached thereto a fluorescent donor
molecule 160. An excitation laser 162 directs a laser beam M toward
detector tip 156 as detector tip 156 is scanned along the length of
selected protein molecule 10. Laser beam M has a wavelength that
excites fluorescent donor molecule 160 to produce emitted radiation
.lambda..sub.160 of a wavelength that will excite a chosen tagged
amino acid residue, such as tagged amino acid residue C shown in
various of protein molecules 10 on support 152. As donor molecule
160 on detector tip 156 is brought in an excited condition into
proximity with amino acid residues C with fluorescent tags, emitted
radiation .lambda..sub.160 excites the tags. The excitation of the
tags on each amino acid residue C in selected protein molecule 10
causes detector tip 156 to vibrate, indicating the positions of
each respective of the amino acid residues C along selected protein
molecule 10.
[0141] Alternatively, the fluorescent donor molecule 160 on the
detector tip 156 of the atomic force microscope 150 could be chosen
so as to emit a wavelength of electromagnetic radiation that
excites tryptophan amino acid residues of a protein molecule or
that excites fluorescent tags on each of another type of amino acid
residue of the protein molecule.
[0142] A third embodiment of an apparatus that is useful for
isolating and characterizing protein molecules according to
teachings of the invention is shown in FIGS. 17 and 18.
[0143] FIG. 17 schematically illustrates an apparatus including a
separation plate 170, a source S of electromagnetic radiation
located on and directed toward a first side 172 of separation plate
170, and a detector 15 located on and facing an opposite, second
side 174 of separation plate 170.
[0144] Separation plate 170 is a thin, planar structure with
separation apertures 176 formed therethrough. Protein molecules 10
are propelled in solution or gel through apertures 176 by way of an
electric field, a technique which is referred to in the art as
electrophoresis. Each aperture 176 has a diameter D sized to permit
a single linear protein molecule 10 to travel therethrough. For
example, diameter D can be about 1-10 nm. The speed at which
protein molecules 10 travel through apertures 176 depends on the
applied electric field. Adjacent apertures 176 are spaced apart
from one another so as to allow for optical resolution
therebetween. For example, adjacent apertures 176 can be spaced
about 1, 10, or 100 .mu.m apart from each other. Separation plate
170 is formed from a material, such as silicon or plastic, that is
opaque to visible wavelengths of electromagnetic radiation and that
can be micromachined or otherwise modified by known processes to
fabricate apertures 176 of desired diameter and spacing.
[0145] Source S emits electromagnetic excitation radiation 178 of
wavelengths that will excite tryptophan amino acid residues or
fluorescent tags on each of one or more other types of amino acid
residues of protein molecules 10 passing through apertures 176. For
example, the lysine and cysteine amino acid residues of protein
molecules 10 could be labeled with different fluorescent tags and
source S could emit a broad range of wavelengths of electromagnetic
radiation to produce the spectrum depicted in FIG. 8.
[0146] Detector 15 is positioned to detect emitted radiation from
amino acid residues of protein molecule 10 as protein molecule 10
exits specific aperture 18 among the several apertures 176 on
second side 174 of separation plate 170. Electromagnetic radiation
maybe focused on detector 15 by way of an optical lens. Known
apparatus that detect electromagnetic radiation, such as a charge
coupled device (hereinafter "CCD") or an avalanche photodiode
array, can be employed as detector 15. Detector 15 can
simultaneously detect fluorescence emitted from amino acid residues
or tags on the amino acid residues of different protein molecules
10. A processor 16 receives signals from detector 15 and generates
data about the particular type of radiation detected.
[0147] FIG. 18 illustrates the phenomenon of "near field
excitation" that occurs as protein molecules 10 that are exposed to
electromagnetic radiation from source S on first side 172 of
separation plate 170 travel through aperture 176. Diameter D is
smaller than the wavelengths of excitation radiation 178 from
source S and emitted radiation from stimulated amino acid residues
W or fluorescent tags on amino acid residues K and C. Thus,
excitation radiation and emitted radiation on first side 172 of
separation plate 170 does not pass through aperture 176. As
stimulated amino acid residue W or a stimulated tag on amino acid
residue K or C exits aperture 176 on second side 174 of separation
plate 170, however, amino acid residue W or the fluorescent tag
remains excited by radiation from source S until leaving a
substantially conical volume having a diameter D and an approximate
height D. This near field excitation, the emitted radiation on
second side 174 of separation plate 170, is detected by detector
15. The signals from detector 15 are then characterized by
processor 16 as representing a specific type of amino acid residue
which, when taken along with the speed at which a protein molecule
travels through aperture 176, is located at a particular position
along the length of protein molecule 10.
[0148] A fourth embodiment of apparatus that isolates and
characterizes protein molecules in accordance with teachings of the
present invention is illustrated in FIGS. 19-27.
[0149] FIG. 19 depicts an isoelectric focusing gel 180 used to
isolate different types of protein molecules in a sample on the
basis of the relative ratio of positively charged regions to
negatively charged regions of each type of protein molecule. For
each type of protein molecule, this ratio is referred to as the
"isoelectric point." The process of isolating protein molecules on
the basis of isoelectric points is referred to as "isoelectric
focusing."
[0150] Prior to being introduced into isoelectric focusing gel 180,
which has a web-like or matrix structure, the protein molecules are
linearized to facilitate the travel of the protein molecules
through the web of isoelectric focusing gel 180. The protein
molecules can be linearized with known chemicals, such as those
discussed above in reference to FIG. 9. When SDS, a negatively
charged, or anionic, detergent is used to linearize the protein
molecules, each of the protein molecules is given a net negative
charge. The protein molecules can also be labeled with tags, in the
same manner described above in reference to FIG. 9. Alternatively,
the protein molecules can be labeled with tags after different
types of protein molecules have been separated from each other.
[0151] Once the linearized protein molecules have been introduced
into isoelectric focusing gel 180, isoelectric focusing gel 180 is
placed in a pH gradient 182. Each protein molecule in isoelectric
focusing gel 180 migrates in the direction of arrow A along the
length of isoelectric focusing gel 180 to a pH that equals the
isoelectric point of that protein molecule.
[0152] Isoelectric focusing can be used to isolate different types
of protein molecules alone or in combination with other separation
techniques. Typically, isoelectric focusing is the first step in a
two-step separation process that is referred to as
"two-dimensional" separation.
[0153] FIG. 20 illustrates the second step of two-dimensional
separation, gel electrophoresis. In gel electrophoresis, native or
linearized protein molecules are introduced into an electrophoresis
gel 184. As depicted, the protein molecules that were previously
separated in isoelectric focusing gel 180 are introduced into
electrophoresis gel 184 at an edge 186 thereof. Like isoelectric
focusing gel 180, electrophoresis gel 184 has a web-like structure.
The passageways through electrophoresis gel 184 are, however, much
smaller than the passageways through isoelectric focusing gel 180
so that the linearized protein molecules traveling through
electrophoresis gel 184 are separated on the basis of size.
[0154] As an electric field 188 is applied to electrophoresis gel
184, the linearized protein molecules travel through
electrophoresis gel 184 in the direction of arrow A. Smaller
protein molecules travel more quickly than larger protein molecules
through the passageways of electrophoresis gel 184.
[0155] FIG. 21 illustrates an electrophoresis gel 184 having
separate bands 190 of different types of protein molecules therein.
Next, as shown in FIG. 22, the protein molecules in bands 190 are
transferred to a membrane 192 of a material such as a
nitrocellulose filter or polyvinylidene difluoride, which is also
referred to as "PVDF," or to another known solid support, such as a
vinyl or nylon support, for further testing, a process referred to
in the art as "blotting." FIG. 23 illustrates membrane 192 and the
bands 194 of different types of protein molecules thereon. Each of
bands 194 corresponds to a similarly located band 190 of
electrophoresis gel 184 in FIG. 21.
[0156] The amino acid residues of the protein molecules in one or
more of bands 194 can be labeled after being isolated on
isoelectric focusing gel 180 and electrophoresis gel 184 and
following the transfer of the protein molecules from bands 190 on
electrophoresis gel 184 to membrane 192. The different types of
protein molecules separated on membrane 192 are then characterized
or identified and compared with one another.
[0157] As depicted in FIG. 24, when the protein molecules are
labeled with fluorescent tags, electromagnetic excitation radiation
196 of one or more appropriate wavelengths can be directed from a
source S toward membrane 192. As the tryptophan amino acid residues
or different tags on one or more different types of amino residues
are stimulated by excitation radiation 196, each of the tryptophan
amino acid residues or tags emit radiation of a distinct wavelength
range, in a similar manner to tryptophan amino acid residue W and
the tags on amino acid residues K and C depicted in FIG. 8. The
different ranges of wavelengths of emitted radiation can be
separated from each other and from excitation radiation 196 by way
of different optical filters 198, 200, 202 that permit only
specific ranges of wavelengths of emitted radiation from amino acid
residues W or from tags on amino acid residues K or C to pass
therethrough.
[0158] When one optical filter 198 is used, the intensity of
emitted radiation from a single type of amino acid residue or from
the tags on a single type of amino acid residue can be detected, in
this case, by a camera 204. Camera 204 can be a digital camera that
creates processable signals representative of the intensities of
emitted radiation of different wavelengths or an optical
camera.
[0159] FIGS. 25, 26, and 27 each illustrate a picture 206, 208, 210
of membrane 192 taken through a single optical filter 198, 200,
202, respectively. The intensity of emitted radiation of each band
194 in each wavelength range represents the number of a particular
type of amino acid residue in the protein molecule isolated in that
band 194 and provides a fingerprint constituent of the type of
protein molecule in that band 194.
[0160] As illustrated, the protein molecules of some bands 194a and
194b do not give off a particular wavelength of emitted radiation.
Bands 194a do not appear in picture 206 of FIG. 24 and band 194b
does not appear in picture 210 of FIG. 27.
[0161] FIGS. 28-33 depict a fifth embodiment of apparatus for
isolating and characterizing a protein molecule. In the fifth
embodiment, certain amino acid residues of protein molecules in a
mixed sample are labeled with fluorescent tags in the manner
described in reference to FIG. 9. The protein molecules of the
mixed sample can optionally be linearized. The protein molecules in
the mixed sample are separated from one another by diluting the
sample. A drop 220 of the diluted sample is then placed on a
microscope cover slip 222, as shown in FIG. 28.
[0162] A microscope slide 224 with a recess 226 formed in a surface
thereof is inverted over droplet 220 to enclose droplet 220 within
recess 226 between microscope slide 224 and cover slip 222. FIG. 29
illustrates a microscope slide 224 prepared in this manner.
[0163] In FIG. 30, a fluorescence microscope 230 is used to detect
the fluorescence of the tryptophan amino acid residues and of the
fluorescent tags on amino acid residues of the protein molecules in
droplet 220. Fluorescence microscope 230 has a source S of
excitation radiation 232 directed toward droplet 220 held by
microscope slide 224. Source S excites tryptophan amino acid
residues and fluorescent tags on one or more other amino acid
residues in the same manner as that depicted in FIG. 8.
[0164] Lens 234 of fluorescence microscope 230 magnifies emitted
radiation from tryptophan amino acid residues and from the
fluorescent tags. The magnified emitted radiation from the
tryptophan amino acid residues and the fluorescent tags on one or
more other types of amino acid residues of the protein molecules in
droplet 220 then passes through an optical filter 236, 238, 240
that permits only a specific range of wavelengths of emitted
radiation from tryptophan amino acid residues or from the tags on
other types of amino acid residues to pass therethrough. Optical
filters 236, 238, and 240 screen out unwanted wavelengths of
emitted radiation. When one optical filter 236 is used, the
intensity of emitted radiation from a single type of amino acid
residue or from the tags on a single type of amino acid residue can
be detected, in this case, by a camera 204 or visually through an
eyepiece 242 of fluorescence microscope 230.
[0165] FIGS. 31-33 illustrate different fields of view of a portion
of droplet 220 through fluorescence microscope 230. FIG. 31 depicts
a field of view 244 through optical filter 236. FIG. 32 depicts a
field of view 246 through optical filter 238. FIG. 33 depicts a
field of view 248 through optical filter 240. As illustrated,
protein molecules 10a emit radiation of some wavelengths and can,
therefore, be seen in fields of view 246 and 248, but not in field
of view 244. The intensities of emitted radiation from other
protein molecules 10b also differ as protein molecules 10b are
visualized through different filters. For example, the intensity of
emitted radiation of a first wavelength from protein molecule 10b
is greater in field of view 244 of FIG. 31 than the intensity of
emitted radiation of a second wavelength from protein molecule 10b
visualized in field of view 246 of FIG. 32 and than the intensity
of emitted radiation of a third wavelength from protein molecule
10b in field of view 248 of FIG. 33, where protein molecule 10b
does not appear to give off any emitted radiation of the third
wavelength. Protein molecule 10c appears in each field of view 244,
246, 248.
[0166] The invention maybe embodied in other specific forms without
departing from its spirit or essential characteristics. The
described embodiments are to be considered in all respects only as
illustrative and not restrictive. The scope of the invention is,
therefore, indicated by the appended claims rather than by the
foregoing description. All changes which come within the meaning
and range of equivalency of the claims are to be embraced within
their scope.
* * * * *