U.S. patent application number 10/899647 was filed with the patent office on 2004-12-30 for protein separation by electrophoresis.
This patent application is currently assigned to Target Discovery, Inc.. Invention is credited to Hall, Michael P., Petesch, Robert, Schneider, Luke V..
Application Number | 20040262160 10/899647 |
Document ID | / |
Family ID | 26828280 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040262160 |
Kind Code |
A1 |
Schneider, Luke V. ; et
al. |
December 30, 2004 |
Protein separation by electrophoresis
Abstract
The present method provides methods and apparatus for separating
proteins using a series of electrophoretic methods that utilize
controlled fractionation and labeling techniques to resolve
mixtures of proteins. The samples for each electrophoretic method
other than the initial method, contain only a subset of proteins
resolved in the preceding method. The methods can be used in a
variety of different applications including, creating proteomic
databases, comparative expression studies, diagnostics, structure
activity relationships and metabolic engineering
investigations.
Inventors: |
Schneider, Luke V.; (Half
Moon Bay, CA) ; Hall, Michael P.; (San Carlos,
CA) ; Petesch, Robert; (Newark, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Target Discovery, Inc.
Palo Alto
CA
|
Family ID: |
26828280 |
Appl. No.: |
10/899647 |
Filed: |
July 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10899647 |
Jul 26, 2004 |
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10341990 |
Jan 13, 2003 |
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10341990 |
Jan 13, 2003 |
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09513486 |
Feb 25, 2000 |
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6537432 |
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60130238 |
Apr 20, 1999 |
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Current U.S.
Class: |
204/451 |
Current CPC
Class: |
G01N 27/44795 20130101;
G01N 33/6848 20130101; G01N 27/44773 20130101; G01N 27/44726
20130101; G01N 27/44782 20130101; Y10T 436/24 20150115; Y10T
436/25125 20150115 |
Class at
Publication: |
204/451 |
International
Class: |
G01L 001/20; G01L
009/18; G01N 027/26 |
Claims
1-61. (Cancelled)
62. A method for separating a plurality of proteins, comprising
performing a capillary isoelectric focusing electrophoresis (CIEF)
or capillary zone electrophoresis (CZE) method with a sample
containing the plurality of proteins, wherein the CIEF method or
CZE method is conducted under conditions such that electroosmotic
flow (EOF) is less than or equal to 0.5.times.10-6
cm.sup.2/V-s.
63. The method of claim 62, wherein the method comprises performing
a CIEF method under conditions such that electroosmotic flow (EOF)
is less than or equal to 0.5.times.10.sup.-6 cm.sup.2/V-s.
64. The method of claim 62, wherein the method comprises performing
a CZE method under conditions such that electroosmotic flow (EOF)
is less than or equal to 0.5.times.10.sup.-6 cm.sup.2/V-s.
65. The method of claim 62, further comprising detecting at least
one protein separated by the CIEF or CZE method.
66. The method of claim 65, further comprising analyzing the at
least one protein to determine a chemical or physical
characteristic of the at least one protein.
67. The method of claim 66, wherein the chemical or physical
characteristic is selected from the group consisting of molecular
weight, complete or partial amino acid sequence, isoelectric point,
relative or absolute abundance and combinations of the
foregoing.
68. The method of claim 66, wherein analyzing is conducted by mass
spectrometry.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application 60/130,238 filed Apr. 20, 1999. This application is
also related to U.S. provisional application 60/075,715 filed Feb.
24, 1998, copending U.S. patent application Ser. No. ______, filed
Feb. 25, 2000, entitled "Methods for Protein Sequencing," and
having attorney docket number 020444-000300US, and copending U.S.
application Ser. No. ______, filed Feb. 25, 2000, entitled
"Polypeptide Fingerprinting Methods and Bioinformatics Database
System," and having attorney docket number 020444-000100US. All of
these applications are incorporated by reference in their entirety
for all purposes.
FIELD OF THE INVENTION
[0002] This invention relates to the field of protein separation
and proteomics.
BACKGROUND OF THE INVENTION
[0003] A goal of genomics research and differential gene expression
analysis is to develop correlations between gene expression and
particular cellular states (e.g., disease states, particular
developmental stages, states resulting from exposure to certain
environmental stimuli and states associated with therapeutic
treatments). Such correlations have the potential to provide
significant insight into the mechanism of disease, cellular
development and differentiation, as well as in the identification
of new therapeutics, drug targets and/or disease markers.
Correlations of patterns of gene expression can also be used to
provide similar insights into disease and organism metabolism that
can be used to speed the development of agricultural products,
transgenic species, and for metabolic engineering of organisms to
increase bioproduct yields or desirable metabolic activities.
[0004] Many functional genomic studies focus on changes in mRNA
levels as being indicative of a cellular response to a particular
condition or state. Recent research, however, has demonstrated that
often there is a poor correlation between gene expression as
measured by mRNA levels and actual active gene product formed
(i.e., protein encoded by the mRNA). This finding is not surprising
since many factors-including differences in translational
efficiency, turnover rates, extracellular expression or
compartmentalization, and post-translational modification affect
protein levels independently of transcriptional controls. Thus, the
evidence indicates that functional genomics is best accomplished by
measuring actual protein levels (i.e., utilizing proteomic methods)
rather than with nucleic acid based methods. The successful use of
proteins for functional genomic analyses, however, requires
reproducible quantification of individual proteins expressed in
cell or tissue samples.
[0005] Two-dimensional (2-D) gel electrophoresis is currently the
most widely adopted method for separating individual proteins
isolated from cell or tissue samples [5, 6, 7]. Evidence for this
is seen in the proliferation (more than 20) of protein gel image
databases, such as the Protein-Disease Database maintained by the
NIH [8]. These databases provide images of reference 2-D gels to
assist in the identification of proteins in gels prepared from
various tissues.
[0006] Capillary electrophoresis (CE) is a different type of
electrophoresis, and involves resolving components in a mixture
within a capillary to which an electric field is applied. The
capillary used to conduct electrophoresis is filled with an
electrolyte and a sample introduced into one end of the capillary
using various methods such as hydrodynamic pressure,
electroosmotically-induced flow, and electrokinetic transport. The
ends of the capillary are then placed in contact with an anode
solution and a cathode solution and a voltage applied across the
capillary. Positively charged ions are attracted towards the
cathode, whereas negatively charged ions are attracted to the
anode. Species with the highest mobility travel the fastest through
the capillary matrix. However, the order of elution of each
species, and even from which end of the capillary a species elutes,
depends on its apparent mobility. Apparent mobility is the sum of a
species electrophoretic mobility in the electrophoretic matrix and
the mobility of the electrophoretic matrix itself relative to the
capillary. The electrophoretic matrix may be mobilized by
hydrodynamic pressure gradients across the capillary or by
electroosmotically-induced flow (electroosmotic flow).
[0007] A number of different electrophoretic methods exist.
Capillary isoelectric focusing (CIEF) involves separating analytes
(such as proteins) within a pH gradient according to the
isoelectric point (i.e., the pH at which the analyte has no net
charge) of the analytes. A second method, capillary zone
electrophoresis (CZE) fractionates analytes on the basis of their
intrinsic charge-to-mass ratio. Capillary gel electrophoresis (CGE)
is designed to separate proteins according to their molecular
weight. (For reviews of electrophoresis generally, and CIEF and CZE
specifically, see, e.g., Palmieri, R. and Nolan, J. A., "Protein
Capillary Electrophoresis: Theoretical and Experimental
Considerations for Methods Development," in CRC Handbook of
Capillary Electrophoresis: A Practical Approach, CRC Press, chapter
13, pp. 325-368 (1994); Kilar, F., "Isoelectric Focusing in
Capillaries," in CRC Handbook of Capillary Electrophoresis: A
Practical Approach, CRC Press, chapter 4, pp. 95-109 (1994); and
McCormick, R. M., "Capillary Zone Electrophoresis of Peptides," in
CRC Handbook of Capillary Electrophoresis: A Practical Approach,
CRC Press, chapter 12, pp. 287-323 (1994). All of these references
are incorporated by reference in their entirety for all
purposes).
[0008] While 2-D gel electrophoresis is widely practiced, several
limitations restrict its utility in functional genomics research.
First, because 2-D gels are limited to spatial resolution, it is
difficult to resolve the large number of proteins that are
expressed in the average cell (1000 to 10,000 proteins). High
abundance proteins can distort carrier ampholyte gradients in
capillary isoelectric focusing electrophoresis and result in
crowding in the gel matrix of size sieving electrophoretic methods
(e.g., the second dimension of 2-D gel electrophoresis and CGE),
thus causing irreproducibility in the spatial pattern of resolved
proteins [20, 21 and 22]. High abundance proteins can also
precipitate in a gel and cause streaking of fractionated proteins
[20]. Variations in the crosslinking density and electric field
strength in cast gels can further distort the spatial pattern of
resolved proteins [23, 24]. Another problem is the inability to
resolve low abundance proteins neighboring high abundance proteins
in a gel because of the high staining background and limited
dynamic range of gel staining and imaging techniques [25, 22].
Limitations with staining also make it difficult to obtain
reproducible and quantifiable protein concentration values, with
average standard variations in relative protein abundance between
replicate 2-D gels reported to be 20% and as high as 45% [4]. In
some recent experiments, for example, investigators were only able
to match 62% of the spots formed on 3-7 gels run under similar
conditions [21; see also 28, 29]. Additionally, many proteins are
not soluble in buffers compatible with acrylamide gels, or fail to
enter the gel efficiently because of their high molecular weight
[26, 27].
SUMMARY OF THE INVENTION
[0009] The present invention provides a variety of electrophoretic
methods and apparatus for separating mixtures of proteins. The
methods involve conducting multiple capillary electrophoresis
methods in series, wherein samples for each method other than the
initial method contain only a subset of the proteins from the
preceding step (e.g., from fractions containing resolved protein
from the preceding method). By using a variety of techniques to
control elution during electrophoresis, the methods are capable of
resolving proteins in even complex mixtures such as obtained from
tissues and native cells. Utilizing various labeling schemes and
detection methods, certain methods can provide quantitative
information on the amount of each of the separated proteins. Such
information can be used in the development of protein databases in
which proteins expressed under certain conditions are characterized
and catalogued. Comparative studies to identify proteins that are
differentially expressed between different types of cells or
tissues can also be conducted with the methods of the present
invention. The methods can also be used in diagnostic, structure
activity and metabolic engineering studies.
[0010] In general, the methods involve performing a plurality of
electrophoretic methods in series. Each method in the series
includes electrophoresing a sample containing multiple proteins to
obtain a plurality of resolved proteins. The sample that is
electrophoresed contains only a subset of the plurality of resolved
proteins from the immediately preceding method in the series
(except the first method of the series in which the sample is the
initial sample that contains all the proteins). The resolved
proteins from the final electrophoretic method are then detected
using various techniques.
[0011] The electrophoretic methods typically are capillary
electrophoresis methods, such as capillary isoelectric focusing
electrophoresis (CIEF), capillary zone electrophoresis (CZE) and
capillary gel electrophoresis (CGE), although the methods are
amenable to other capillary electrophoresis methods as well. The
particular order of the methods can vary. Typically, the methods
utilize combinations of electrophoretic methods which separate
proteins on the basis of different characteristics (e.g., size,
charge, isoelectric point).
[0012] In certain methods, the proteins are labeled to more easily
detect the resolved proteins, to alter the charge of the proteins,
to facilitate their separation, and/or to increase the
signal-to-noise ratio. Labeling also enables certain methods to be
conducted such that the resolved proteins obtained from the final
electrophoretic method are quantitated. Quantitation allows the
relative abundance of proteins within a sample, or within different
samples, to be determined. In certain methods, the time at which
proteins are labeled is selected to precede electrophoresis by
capillary zone electrophoresis. By selectively labeling certain
residues, resolution of proteins during capillary zone
electrophoresis can be increased.
[0013] Resolution, quantitation and reproducibility are enhanced by
utilizing a variety of techniques to control elution of proteins
during an electrophoretic method. The particular elution technique
employed depends in part upon the particular electrophoretic
method. However, in general, hydrodynamic, salt mobilization, pH
mobilization and electroosmotic flow are utilized to controllably
elute resolved proteins at the end of each electrophoretic
separation.
[0014] Some methods provide for additional analysis after the
electrophoretic separation. The type of analysis can vary and
include, for example, infra-red spectroscopy, nuclear magnetic
resonance spectroscopy, UV/VIS spectroscopy, fluorescence
spectroscopy, and complete or partial sequencing. In certain
methods, proteins in the final fractions are further analyzed by
mass spectroscopy to determine at least a partial sequence for each
of the resolved proteins (i.e., to determine a protein sequence
tag).
[0015] Thus, certain other methods involve performing one or more
capillary electrophoretic methods, each of the one or more methods
involving: (i) electrophoresing a sample containing multiple
proteins within an electrophoretic medium contained within a
capillary, and (ii) withdrawing and collecting multiple fractions,
each fraction containing proteins resolved during the
electrophoresing step. Each method in the series is conducted with
a sample from a fraction collected in the preceding electrophoretic
method, except the first electrophoretic method which is conducted
with a sample containing the original mixture of proteins. The
proteins are labeled prior to conducting the last electrophoretic
method. Either the proteins in the initial sample are labeled
(i.e., labeling precedes all the electrophoretic separations), or
the proteins contained in fractions collected are labeled prior to
the last electrophoretic method. The final electrophoretic method
is performed, and resolved protein within, or withdrawn from, the
capillary utilized to conduct the final method is detected with a
detector. Hence, the detector is adapted to detect resolved protein
within the capillary used in the final method or is connected in
line with the capillary to detect resolved proteins as they elute
from the capillary. In some instances, the detected proteins are
quantitated and further analyzed by mass spectroscopy to determine
their relative abundance and/or to establish a protein sequence tag
for each resolved protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic representation of one example of an
electrophoretic system that can be utilized with certain methods of
the invention.
[0017] FIG. 2A is a schematic representation of some of the major
elements of an electrophoretic system utilized in conducting
certain electrophoretic methods of the invention.
[0018] FIG. 2B is a cross-sectional view of a capillary showing the
orientation of a porous plug inserted into the capillary to control
electroosmotic flow in certain methods of the invention.
[0019] FIGS. 3A and 3B are top-views of certain elements of
microfluidic devices that can be utilized to'conduct certain
electrophoretic methods of the invention.
[0020] FIG. 4 is an electropherogram for a sample containing five
unlabeled proteins (hen egg white conalbumin, bovine serum albumin,
bovine carbonic anhydrase II, carbonic anhydrase II, rabbit muscle
GAPDH, and bovine ribonuclease A) as obtained following
electrophoresis by capillary zone electrophoresis. Absorbance was
monitored at 214 nm. Under the conditions of this particular
experiment (see Example 1) in which the proteins were unlabeled,
the proteins were not resolved.
[0021] FIG. 5 is a plot of electrophoretic mobility for each of the
five proteins listed in FIG. 4 under the same electrophoresis
conditions as described in FIG. 4.
[0022] FIG. 6 is a plot showing the correlations between
electrophoretic mobility and the predicted mass-to-charge ratio of
the proteins at pH 4.0.
[0023] FIG. 7 is an electropherogram obtained during separation of
a sample containing five sulfophenylisothiocyanate-labeled proteins
(hen egg white conalbumin, bovine serum albumin, bovine carbonic
anhydrase II, carbonic anhydrase II, rabbit muscle GAPDH, and
bovine ribonuclease A) as obtained following electrophoresis by
capillary zone electrophoresis. Absorbance was monitored at 214 nm.
Under the conditions of this particular experiment (see Example 2)
in which the proteins were labeled, the labeled proteins were
partially resolved.
[0024] FIG. 8 is an electropherogram obtained during separation of
a sample containing the proteins hen white conalbumin, bovine serum
albumin, and bovine carbonic anhydrase II, by CIEF.
[0025] FIG. 9 is an electropherogram of a fraction (fraction F)
obtained from the separation by CIEF shown in FIG. 7.
[0026] FIG. 10 is an electropherogram of a fraction (fraction G)
obtained from the separation by CIEF shown in FIG. 7.
DETAILED DESCRIPTION
[0027] I. Overview
[0028] The present invention provides methods and apparatus for
achieving the separation of proteins, including significant
resolution of proteins in complex mixtures from native cell and
tissue samples. The invention is based in part upon the recognition
that multidimensional electrophoretic methods involving multiple
(typically different) electrophoretic methods performed in series
utilizing controlled fractionation techniques to obtain defined
fractions can be used to achieve high resolution of proteins.
Labeling and detection steps can be included to increase
sensitivity, alter the separation coordinates of the proteins, and
to obtain accurate and reproducible quantitative information about
the resolved proteins. Typically, the electrophoretic methods are
capillary electrophoresis methods, particularly combinations of
capillary isoelectric focusing (CIEF), capillary zone
electrophoresis (CZE) and capillary gel electrophoresis (CGE).
[0029] Several features enable methods to be performed in a
controlled and reproducible fashion. For example, once proteins
have had an opportunity to fractionate within the electrophoretic
medium contained within a capillary, elution conditions are
tailored so that separated proteins are eluted in a controlled
fashion to yield defined fractions in which the proteins contained
within a fraction fall within a certain pH range, electrophoretic
mobility range, or molecular weight range, for example. In certain
methods, proteins are labeled at a selected stage of the separation
process and the labeled proteins detected using a detector.
Labeling enables proteins present at low concentration to more
easily be detected and enhances reproducibility by increasing
signal-to-noise ratios. The detector can be used to detect proteins
as separated within an electrophoretic cavity or after they are
eluted from the cavity. The combination of labeling and detection
also enables separated proteins to be quantified. The combination
of labeling and separation can alter the net charge or solubility
of the proteins causing a change in their separation coordinates,
for example, their separation order, the fraction in which they are
collected, and elution time.
[0030] If additional information is desired, the methods can be
expanded to include further analysis by techniques besides
electrophoresis. For example, in certain methods, fractions
collected from the final electrophoretic method are individually
analyzed by mass spectroscopy to obtain additional information,
such as molecular weight and partial sequence.
[0031] Quantitative detection and the ability to automate the
methods means that the methods are amenable to a variety of
screening, comparative and diagnostic studies. For example, the
methods can be utilized to develop comparative protein expression
data. Such comparative studies can be utilized to identify markers
of specific diseases, potential targets for pharmaceuticals and/or
drug candidates. Once markers that are selectively expressed in
certain disease states, for example, are identified, the methods of
the invention have utility in diagnostic applications. The methods
of the invention can also be utilized to develop a protein database
that includes, for example, separation coordinates, isoelectric
points, apparent molecular weights and relative abundance
information for proteins in different cells, tissues or states. The
methods also find utility in studies on structure/activity
relationships and in metabolic engineering investigations in which
one genetically modifies a certain gene and then determines what
effects such a modification has on cellular protein expression.
[0032] II. Separation Methods
[0033] A. General
[0034] The methods of the present invention utilize a combination
of electrophoretic methods conducted in series to resolve mixtures
of proteins. The methods are said to be conducted in series because
the sample(s) electrophoresed in each method are from solutions or
fractions containing proteins electrophoresed in the preceding
method, with the exception of the sample electrophoresed in the
initial electrophoretic method. As used herein, the terms protein,
peptide and polypeptide are used interchangeably and refer to a
polymer of amino acid residues. The term also applies to amino acid
polymers in which one or more amino acids are chemical analogues of
corresponding naturally-occurring amino acids, including amino
acids which are modified by post-translational processes (e.g.,
glycosylation and phosphorylation).
[0035] The series of electrophoretic methods are typically
conducted in such a way that proteins in an applied sample for each
electrophoretic method of the series are isolated or resolved
physically, temporally or spacially to form a plurality of
fractions each of which include only a subset of proteins of the
applied sample. Thus, a fraction refers to a protein or mixture of
proteins that are resolved physically, temporally or spacially from
other proteins in a sample subjected to electrophoresis. Resolved
proteins can refer to a single species or a mixture of proteins
that are separated from other proteins during an electrophoretic
method. As just noted, samples in the various electrophoretic
methods are obtained from such fractions, with the exception of the
first electrophoretic method in which the sample is the original
sample containing all the proteins to be separated.
[0036] Typically, these multiple electrophoretic methods in the
series separate proteins according to different characteristics.
For example, one method can separate proteins on the basis of
isoelectric points (e.g., capillary isoelectric focusing
electrophoresis), other methods can separate proteins on the basis
of their intrinsic or induced (through the application of a label
to certain ionizable amino acid residues) charge-to-mass ratio at
any given pH (e.g., capillary zone electrophoresis), whereas other
methods separate according to the size of the proteins (e.g.,
capillary gel electrophoresis). Such approaches that separate
proteins through a series of electrophoretic methods are referred
to herein as "multidimensional" electrophoretic methods, wherein
each particular electrophoretic method constitutes a
"dimension."
[0037] Apparatus used to conduct various electrophoretic methods
are known in the art. In general, however, and as shown in FIG. 2A,
the basic configuration of a typical capillary electrophoretic
system utilized in certain methods of the invention includes a
capillary 8 having two ends 10, 12. One end 10 is in contact with
an anode solution or anolyte 14 contained in an anode reservoir 18
and the other end 12 is in contact with a cathode solution or
catholyte 16 in a cathode reservoir 20. One electrode (the anode)
22 is positioned to be in electrical communication with the anode
solution 14 and a second electrode 24 is positioned to be in
electrical communication with the cathode solution 16. The cavity
26 of the capillary 8 is filled with an electrophoretic medium,
which in some instances can include a polymer matrix. As used
herein, the term anode refers to the positively charged electrode.
Thus, negatively charged species move through the electrophoretic
medium toward the anode. The term cathode refers to the negatively
charged electrode; positively charged species migrate toward this
electrode. The anolyte is the solution in which the anode is
immersed and the catholyte is the solution in which the cathode is
immersed.
[0038] Sample is introduced into the capillary 8 via an inlet 28,
and the protein components therein resolved as an electrical field
is applied between the two electrodes 22, 24 by a power source 32
and the proteins separate within the electrophoretic medium
contained within the separation cavity 26. Protein components can
be controllably eluted from the capillary via outlet 30 by
controlling various parameters such as electroosmotic flow (see
infra) and/or by changing the composition of one or both of the
reservoir solutions (e.g., adjusting the pH or salt concentration).
Typically, the inlet 28 and the outlet 30 are simply portions of
the capillary formed to allow facile insertion into a container
containing sample, anolyte or catholyte.
[0039] The term "capillary" as used in reference to the
electrophoretic device in which electrophoresis is carried out in
the methods of the invention is used for the sake of convenience.
The term should not be construed to limit the particular shape of
the cavity or device in which electrophoresis is conducted. In
particular, the cavity need not be cylindrical in shape. The term
"capillary" as used herein with regard to any electrophoretic
method includes other shapes wherein the internal dimensions
between at least one set of opposing faces are approximately 2 to
1000 microns, and more typically 25 to 250 microns. An example of a
non-tubular arrangement that can be used in certain methods of the
invention is the a Hele-Shaw flow cell [67, 68]. Further, the
capillary need not be linear; in some instances, the capillary is
wound into a spiral configuration, for example.
[0040] An example of a system utilized with certain methods of the
invention is illustrated in FIG. 1. This particular example shows a
system in which three electrophoresis methods (initial,
intermediate and final methods) are linked. The particular number
of electrophoretic methods conducted can vary, although the methods
of the invention include at least two electrophoretic methods. Most
typically, the methods utilize two or three electrophoretic
separation methods.
[0041] As can be seen in FIG. 1, an initial sample containing a
plurality of proteins is introduced from sample container 50 into a
first separation cavity of a first capillary 54 via sample inlet 52
utilizing any of a number of methods known in the art. Examples of
suitable methods include, pulling sample into the sample inlet 52
under vacuum (e.g., by pulling a vacuum on the sample outlet) or
pushing sample into the sample inlet 52 by pressurizing the sample
container 50. Electromigration, often referred to as electrokinetic
injection, is another option. Once the initial sample is introduced
into sample inlet 52, the sample is then electrophoresed within the
first separation cavity within the first capillary 54. The first
separation cavity contains a desired electrophoretic medium in
which proteins in the initial sample are at least partially
resolved. Electrophoretic medium containing resolved proteins is
withdrawn from the first cavity, typically out the end of the
separation cavity opposite the end in which sample was introduced,
although other withdrawal sites can be utilized (see infra). The
withdrawn medium travels through outlet 56 and is collected in
separate containers 58 as multiple fractions. As shown in FIG. 1B,
the containers 58 into which fractions are collected are typically
associated with a fraction collection device (a portion of which is
shown 60) capable of automatically advancing a set of containers 58
to collect defined fractions (e.g., fractions of a certain volume
or covering a selected pH range).
[0042] A sample from a fraction collected from the first
electrophoretic method is then withdrawn from one of the plurality
of containers 58, again utilizing techniques such as those
described supra, via a second sample inlet 62. Proteins in the
sample from the fraction can then be further resolved by conducting
an intermediate electrophoretic method (in the example shown in
FIG. 1, the second electrophoretic method). The sample is
introduced into a second capillary 64 via inlet 62 and the proteins
within the sample further separated within the electrophoretic
medium contained within the second separation cavity of the second
capillary 64 and then eluted from the cavity via outlet 66. As with
the first electrophoretic separation, the electrophoretic medium
containing the resolved or partially resolved proteins is collected
as separate fractions within containers 68 typically aligned and
advanced by a second fraction collection device (a portion of which
is shown 70).
[0043] A process similar to the second/intermediate method is
conducted during the final electrophoretic method (the third
electrophoretic separation method shown in FIG. 1). Sample is drawn
via inlet 72 from a container 68 containing a fraction obtained
during the preceding method and is introduced into a third or final
electrophoretic cavity of a third capillary 74 containing a third
electrophoretic medium in which proteins contained in the applied
sample are separated still further yet by electrophoresis. The
third electrophoretic medium containing the further isolated
proteins is subsequently withdrawn through outlet 76.
[0044] As noted above, more than the three electrophoretic methods
shown in FIG. 1 can be performed. Such methods essentially involve
repeating the general steps described for the second/intermediate
electrophoretic separation above one or more times.
[0045] Following the final electrophoretic separation, a variety of
different options for analyzing the resolved proteins are
available. As shown in FIG. 1, withdrawn electrophoretic medium can
be passed through a detector 78 in fluid communication with the
separation cavity of the last capillary 74 to detect the resolved
proteins. The detector 78, or an optional quantifying device
capable of receiving a signal from the detector (not shown), can be
used to quantitate the amount of protein within a certain portion
or fraction of the electrophoretic medium.
[0046] Alternatively, or in addition, fractions can be taken from
the electrophoretic medium exiting the final capillary 74 or the
detector 78 and analyzed by an analyzer 82 using some technique
other than electrophoresis. Examples of such techniques include
various spectroscopic methods (e.g., IR, UV/VIS and NMR) and
various mass spectroscopy methods (e.g., electrospray
ionization-time of flight [ESI-TOF] mass spectroscopy). Mass
spectral data, for example, can be utilized to deduce a partial or
full sequence of the protein(s) (i.e., determine a protein sequence
tag) within a particular fraction. FIG. 1 depicts a situation in
which sample is withdrawn via line 80 (dashed to indicate optional
nature of this step) to another analyzer 82 (e.g., mass
spectrometer).
[0047] A number of other configurations can be utilized. For
example, the capillaries and detector(s) can be fabricated within a
microfluidic chip (see infra).
[0048] The specific elution conditions utilized to withdraw
resolved proteins from the separation cavity depends upon the type
of electrophoretic method conducted and is described more fully
below for each of the electrophoretic methods typically utilized in
the present invention. In general, however, once proteins have been
resolved, the conditions within the separation cavity are adjusted
as necessary (or the initial conditions selected) to achieve
selective or controlled elution of the proteins from the cavity.
For example, elution can be achieved by adding salts to, or
adjusting the pH of, the anode or cathode solution, by regulating
electroosmotic flow, by applying hydrodynamic pressure or
combinations of the foregoing.
[0049] Using the methods of the invention, resolved proteins can be
isolated physically (e.g., placement into different containers such
as illustrated in FIG. 1), spatially (e.g., spread throughout the
electrophoretic medium contained in the separation cavity) and/or
temporally (e.g., controlling elution so different proteins within
a sample elute from the capillary at different times). Thus, the
methods of the invention can separate mixtures of proteins as a
function of the composition of elution buffers and/or time, and are
not limited to the spatial separation of proteins as are certain
traditional two-dimensional (2-D) gel electrophoresis systems.
Instead, with controlled elution, fractions can be collected so
that proteins within a fraction fall within a range of isoelectric,
electrophoretic mobility, or molecular weight values, for example.
Controlled elution of proteins means that methods can be performed
in a reproducible fashion. Such reproducibility is important in
conducting comparative studies and in diagnostic applications, for
example.
[0050] During the elution or withdrawing of resolved proteins,
generally only a portion of the electrophoretic medium containing
the resolved proteins is typically collected in any given fraction.
This contrasts with certain 2-D methods in which a gel containing
all the resolved proteins is exuded from the separation cavity and
the exuded gel containing all the proteins is used to conduct
another electrophoretic separation.
[0051] Spacially, physically or temporally resolved proteins
obtained at the conclusion of one electrophoretic method are then
used as the source of samples for further separation of proteins
contained within the fraction during a subsequent electrophoretic
method. As illustrated in FIG. 1, typically samples from different
resolved fractions are sequentially electrophoresed on the same
capillary. Normally another sample is not applied until the
proteins in the preceding sample are sufficiently withdrawn from
the separation cavity so that there is no overlap of proteins
contained in different fractions. Sequential elution of fractions
through the same column can significantly reduce or eliminate
variations resulting from differences in cross-linking or electric
field strength that can be problematic in certain slab gel
electrophoretic methods. Hence, sequential separation can further
enhance the reproducibility of the methods of the invention. Other
methods, however, can be performed in a parallel format, wherein
samples from different fractions are electrophoresed on separate
capillaries. This approach allows for separations to be completed
more quickly. However, the use of multiple capillaries can increase
the variability in separation conditions, thereby reducing to some
extent reproducibility between different samples.
[0052] In certain methods, proteins are labeled at a selected stage
of the separation process and then detected using the detector.
Labeling enables proteins present at low concentration to more
easily be detected and enhances reproducibility by increasing
signal-to-noise ratios. The detector can be used to detect proteins
as separated within an electrophoretic cavity or after they are
eluted from the cavity. The combination of labeling and detection
also enables separated proteins to be quantified. The point in the
overall method at which labeling is conducted depends in part on
the particular electrophoretic methods being conducted as discussed
more fully below. In general, however, labeling is typically
conducted before a gel capillary electrophoretic separation is
performed; whereas, labeling is normally conducted after capillary
isoelectric focusing is performed rather than before. Labeling can
also be used before a zone capillary electrophoresis separation is
performed as a means to modify the net charge on the proteins and
their relative electrophoretic mobilities.
[0053] As noted above, some of the more commonly used
electrophoretic methods utilized in the present invention are
capillary isoelectric focusing electrophoresis, capillary zone
electrophoresis and capillary gel electrophoresis. Specific issues
regarding the performance of these methods are described in the
following sections.
[0054] B. Capillary Isoelectric Focusing Electrophoresis (CIEF)
[0055] 1. General
[0056] Isoelectric focusing is an electrophoretic method in which
zwitterionic substances such as proteins are separated on the basis
of their isoelectric points (pI). The pI is the pH at which a
zwitterionic species such as a protein has no net charge and
therefore does not move when subjected to an electric field. In the
present invention, proteins can be separated within a pH gradient
generated using ampholytes or other amphoteric substances within an
electric field. A cathode is located at the high pH side of the
gradient and an anode is located at the low pH side of the
gradient. Proteins introduced into the gradient focus within the pH
gradient according to their isoelectric points and then remain
there. General methods for conducting CIEF are described, for
example, by Kilar, F., "Isoelectric Focusing in Capillaries," in
CRC Handbook on Capillary Electrophoresis: A Practical Approach,
CRC Press, Inc., chapter 4, pp. 95-109 (1994); and Schwartz, H.,
and T. Pritchett, "Separation of Proteins and Peptides by Capillary
Electrophoresis: Application to Analytical Biotechnology," Part No.
266923 (Beckman-Coulter, Fullerton, Calif., 1994); Wehr, T.,
Rodriquez-Diaz, R., and Zhu, M., "Capillary Electrophoresis of
Proteins," (Marcel Dekker, NY, 1999), which are incorporated herein
by reference in their entirety.
[0057] 2. System and Solutions
[0058] Because CIEF is primarily an equilibrium technique with low
current densities, capillary heating typically is not a problem.
Therefore, fairly large bore capillaries can be utilized. Suitable
sizes include, but are not limited to, capillaries having internal
diameters of 2-600 .mu.m, although more typically capillaries
having internal diameters of 25-250 .mu.m are utilized. The use of
relatively large bore capillaries means the method can use
relatively high protein loads, which facilitates detection in the
following dimension(s). This feature of CIEF makes the method
well-suited for the initial or one of the early electrophoretic
separations in the series. However, smaller diameter capillaries
enable temperature to be controlled more carefully and, in some
methods, result in improved signal detection (e.g., by laser
induced fluorescence (LIF) detection of fluorescently labeled
proteins).
[0059] The capillaries can have varying lengths. The length
selected depends in part on factors such as the extent of
separation required. Typically, the capillaries are about 10 to 100
cm in length, although somewhat shorter and longer capillaries can
be used. While longer capillaries typically result in better
separations and improved resolution of protein mixtures, longer
capillaries also afford more opportunities for protein-wall
interactions and lower field strength. Consequently, there tends to
be an upper limit on capillary length beyond which resolution may
be lost. Longer capillaries can be of particular use in resolving
low abundance proteins. Further guidance on size and length of
capillaries is set forth, for example, in Palmieri, R. and J. A.
Nolan, "Protein capillary electrophoresis: Theoretical and
experimental considerations for methods development," in: CRC
Handbook of Capillary Electrophoresis: A Practical Approach, Chp.
13, pgs. 325-368 (CRC Press, Boca Raton, 1994).
[0060] Generally, the capillaries are composed of flised silica,
although plastic capillaries and PYREX (i.e., amorphous glass) can
be utilized in certain methods. As noted above, the capillaries do
not need to have a round or tubular shape. Other shapes wherein the
internal dimension between opposing faces is within the general
range set forth in this section can also be utilized.
[0061] A variety of different anode and cathode solutions can be
used. Common solutions include sodium hydroxide as the catholyte
and phosphoric acid as the anolyte. Similarly, a number of
different ampholytes can be utilized to generate the pH gradient,
including numerous commercially available ampholyte solutions
(e.g., BioLyte, Pharmalyte and Servalyte). The selection of
ampholytes and the breadth of the ampholyte gradient can impact the
resolution that is achieved by CIEF methods. Narrow ampholyte
gradients increase the number of theoretical plates in the
separation and can be beneficial for higher resolution-separations
over narrow pI ranges.
[0062] CIEF methods utilized in the separations of the invention
can be conducted in capillaries containing polymeric matrices or in
free solution (i.e., no gel or other polymeric matrix). Polymer
matrices are typically added to slow electroosmotic flow; however,
in some instances, inclusion of polymeric matrices can restrict
movement of larger proteins (see, e.g., Patton, 26). The use of
free solutions is preferable in such cases possibly in combination
with other methods (e.g., capillary coatings, gel plugs, or induced
electric fields) to control the electroosmotic flow.
[0063] 3. Sample Preparation
[0064] Typically protein samples to be electrophoresed by CIEF are
denatured prior to loading the sample into the capillary. This
ensures that the same proteins all have the same charge and thus
identical proteins focus at the same location rather than
potentially at multiple zones within the capillary. Denaturants
(e.g., urea), non- and zwitterionic-surfactants (e.g., IGEPAL
CA-630 or 3-[{3-cholamidopropyl}di- methylammonio]-1-propane
sulfonate) can also be used to suppress protein-wall and/or
protein-protein interactions that can result in protein
precipitation. Another advantage of denaturing the proteins prior
to electrophoresis is that the results can be used in comparisons
with archival data typically obtained under denaturing
conditions.
[0065] A typical denaturing buffer includes urea and a nonionic or
zwitterionic surfactant as denaturants; a reducing agent (e.g.,
dithiothreitol (DTT) or mercaptoethanol) is typically included to
reduce any disulfide bonds present in the proteins. Other
denaturants besides urea that can be used include, but are not
limited to, thiourea and dimethylformamide (DMF). Generally,
guanidine hydrochloride is not utilized as a denaturant because of
the very high ionic strength it imparts to a sample. Exemplary
neutral detergents include polyoxyethylene ethers ("tritons"), such
as nonaethylene glycol octylcyclohexyl ether ("TRITON" X-100),
polyglycol ethers, particularly polyalkylene alkyl phenyl ethers,
such as nonaethylene glycol octylphenyl ether ("NONIDET" P-40 or
IGEPAL CA-630), polyoxyethylene sorbitan esters, such as
polyoxyethylene sorbitan monolaurate ("TWEEN"-20), polyoxyethylene
ethers, such as polyoxyethylene lauryl ether (C.sub.12E.sub.23)
("BRIJ"-35), polyoxyethylene esters, such as 21 stearyl ether
(C.sub.18E.sub.23) ("BRIJ"721),
N,N-bis[3-gluconamido-propyl]cholamide ("BIGCHAP"),
decanoyl-N-methylglucamide, glucosides such as octylglucoside,
3-[{3-cholamidopropyl}dimethylammonio]-1-propane sulfonate and the
like.
[0066] The optimal amount of denaturant and detergent depends on
the particular detergent used. In general the denaturing sample
buffers contain up to 10 M urea (more typically 4-8 M and most
typically 6-8 M). Specific examples of suitable buffers (and
denaturants and nonionic surfactants for inclusion therein) include
those described by Hochstrasser et. al.[5] and O'Farrell[6].
Denaturation is typically advanced by heating for 10 min at
95.degree. C. prior to injection into the capillary. Adjustments in
the denaturing sample buffers are made as necessary to account for
any electroosmotic flow or heating effects that occur (see, e.g.,
Kilar, F., "Isoelectric Focusing in Capillaries," in CRC Handbook
on Capillary Electrophoresis: A Practical Approach, CRC Press,
Inc., chapter 4, pp. 95-109 (1994)).
[0067] The amount of protein within a sample can vary and, as noted
above, depends in part of the size of the capillary used. In
general, the capillary is loaded with 0.1 to 5.0 mg of total
protein. Samples can be spiked with one or more known pI standards
to assess the performance of the method.
[0068] 4. Elution
[0069] A variety of techniques can be utilized to elute or withdraw
electrophoretic medium containing resolved proteins out from the
capillary, but these methods fall into three general categories:
hydrodynamic elution, electroelution and control of electroosmotic
flow.
[0070] a. Hydrodynamic/Pressure Elution
[0071] Hydrodynamic or pressure elution involves applying pressure
(or pulling a vacuum) via an appropriate pump connected with one
end of the capillary (see, e.g. Kilar, F., "Isoelectric Focusing in
Capillaries," in CRC Handbook on Capillary Electrophoresis: A
Practical Approach, CRC Press, Inc., chapter 4, pp. 95-109 (1994)).
However, hydrodynamic elution can cause band broadening and loss of
resolution due to the parabolic flow profile that is formed in the
capillary.
[0072] b. Electroelution
[0073] Electroelution, the other major approach, encompasses a
variety of techniques and in general involves altering the solution
at the anode and/or cathode to change some parameter (e.g., pH,
ionic strength, salt concentration) of the electrophoretic medium
in the separation cavity sufficiently to effect elution.
[0074] i. Salt mobilization
[0075] One electroelution approach involves addition of a salt to
the catholyte or anolyte, the salt having a non-acidic or non-basic
counterion of the same charge as the acidic or basic species within
the reservoir to which the salt is added so that the counterion
migrates from the reservoir into the capillary. Since electrical
neutrality must be maintained within the capillary, the movement of
the counterion into the capillary results in a reduction of the
concentration of protons or hydroxide within the capillary, and
thus the pH is either raised or lowered. The theoretical basis for
this type of mobilization is described by S. Hjerten, J.-L. Liao,
and K. Yao, J. Chromatogr., 387: 127 (1987). For example, if the
catholyte is sodium hydroxide (i.e., the basic species is
hydroxide) then a salt having a negatively charged counterion other
than hydroxide is added, for example sodium chloride. Movement of
chloride ion into the capillary reduces the local concentration of
hydroxide within the capillary, thereby decreasing the pH. As
another example, if the anolyte is phosphoric acid, then a salt
having a counterion other than a proton is added, for example
sodium phosphate. In this instance, movement of sodium ion into the
capillary reduces the local concentration of protons within the
capillary thereby increasing the pH. As the pH is lowered or raised
within regions of the capillary due to the presence of the added
counterion, elution occurs since the ampholytes, and the focused
proteins, migrate to the newly-defined pH regions corresponding to
their isoelectric points. It has been shown that both the type and
concentration of salt used for mobilization has impact on the
resolution of eluted protein peaks [R. Rodriguez-Diaz, M. Zhu, and
T. Wehr, J. Chromatogr. A, 772:145 (1997)]. In particular, the
addition of sodium tetraborate instead of sodium chloride to the
catholyte results in greatly increased resolution of separated
proteins.
[0076] ii. pH Mobilization
[0077] Another technique, referred to herein as "pH mobilization"
can also be utilized to elute proteins during CIEF. In this'
approach, an additive is added to either the anode or cathode
solution to alter the pH of the solution. Unlike salt mobilization,
however, the additive does not contribute a mobile counteribn that
moves into the capillary. Here, the elution occurs as a result of
the pH gradient being redefined by the pH of one or both of the
reservoirs; therefore, proteins with pI's that fall outside of this
redefined pH gradient are eluted into either the anode or cathode
reservoirs. Typically, the technique for cathodic mobilization
would proceed as follows. Once the proteins are focused in an
exemplary pI range of 3-10 using phosphoric acid as the anolyte and
sodium hydroxide as the catholyte, the cathodic capillary end is
immersed into a reservoir containing a solution that has a pH
slightly less than 10, for example 50 mM imidazole (pKa 7) which
has a pH of 9.85. The proteins are then allowed to refocus in the
capillary, recognizable by a stabilization of the current through
the capillary, the pI range now being defined by 3-9.85. Any
proteins with an isoelectric point of 9.85 to 10 are eluted into
the catholyte. The process can be repeated with catholyte
containing a species that reduces the pH to slightly less than
9.85. In a stepwise fashion, the pH can be continued to be reduced
to pH 7, thereby collecting separated proteins in fractions that
span the range of 7-10. At this point, anodic mobilization can
proceed by replacing the anolyte with acids of increasing pKa to
selectively increase the pH from 3 to 7, thereby collecting
fractions in the acidic range (pH 3-7). The number of fractions can
vary depending on the desired fractionation resolution. Typically,
these fractions are defined by differences of 0.05-0.5 pH
units.
[0078] The technique of pH mobilization can be useful for protein
samples containing a high concentration of one or more proteins
that may cause uneven spatial gradients inside the capillary. Using
pH mobilization, only those proteins with isoelectric points below
or above the pI range that is defined by the reservoir pH's are
eluted. This elution would, therefore, be reproducible regardless
of differences in the shape of the capillary pH gradient or the
presence of uneven spatial gradients inside the capillary.
[0079] c. Electroosmotic Flow (EOF)
[0080] Regulating the magnitude of electroosmotic flow (EOF)
significantly affects the preceding electroelution methods (see
supra) and is another means by which resolved proteins can be
selectively withdrawn upon conclusion of an isoelectric focusing
separation. EOF is generated by the ionization of silanol
functionalities on the surface of a silica capillary. Such
ionization results in a layer of protons in the electrophoretic
medium at the surface of the silica capillary. Once an electric
field is applied, the layer of protons essentially constitutes a
positively charged column of fluid which migrates toward the
cathode, thereby causing bulk flow of the electrophoretic medium
within the capillary. Apparent velocity of analytes is equal to the
sum of the electroosmotic flow and their electrophoretic mobility.
Thus, by controlling EOF, one can control or regulate the rate at
which proteins move through the capillary. In CIEF methods,
generally EOF should be controlled to allow proteins within an
injected sample sufficient time to focus before the proteins begin
eluting from the capillary.
[0081] A variety of techniques can be utilized to regulate EOF. One
approach involves coating the walls of capillaries with various
agents. For example, EOF along glass silicate surfaces can be
substantially reduced by silanizing them with a neutral silane
reagent that masks a substantial percentage of surface silanol
groups (e.g., polyacrylamide, polyethylene glycol and polyethylene
oxide). The magnitude of EOF can be further controlled by using
silanizing reagents that include positively r negatively charged
groups. Positively charged coatings can be used to nullify surface
negative charges to give a net surface charge of zero, so that EOF
approaches zero. Coatings with higher positive charge densities can
be used to reverse the direction of EOF for charged surface
materials. This can be useful for slowing the net migration rates
of positively charged sample species. Conversely, negatively
charged coatings can be used to impart to or increase the magnitude
of the negative charge on surfaces, so as to increase the net
migration rates of negatively charged species. Representative
positively charged coatings include trialkoxysilanes with
polyethyleneimine, quaternized polyethyleneimine,
poly(N-ethylaminoacrylamide) and chitosans, for example.
Representative negatively charged coatings include trialkoxysilanes
with carboxylate and sulfonate containing materials such as
poly(methylglutamate) and 2-acrylamido-2-methylpropanesulfonate
polymers, for example. It will be recognized that charged coatings
can also effectively reduce sample adsorption, especially for
samples having the same charge polarity as the coating.
[0082] The separation medium can also include soluble agents for
dynamically coating the walls of the separation cavity, to help
reduce EOF during electrophoresis. Such soluble coating agents
include quaternary ammonium-containing polymers, methyl cellulose
derivatives, cellulose acetate, polyethylene oxide, chitosan,
polyvinyl alcohol, polyethylene glycol, polyethylenimine, and
polyethylene oxide-polypropylene oxide-polyethylene oxide triblock
copolymers, for example. Typically, soluble coating agents are
included at concentrations of about 0.05% to about 4%, and more
typically of about 1% to about 2%.
[0083] EOF and sample absorption can also be adjusted by including
suitable reagents in the separation medium and running buffers. For
example, negative surface charges can be masked by including a
cationic additive in the medium, such as metal amine complexes,
amines and polyamines such as propylamine, triethylamine,
tripropylamine, triethanolamine, putrescine, spermine,
1,3-diaminopropane, morpholine, and the like. Zwitterionic species
comprising both negatively and positively charged groups that are
isoelectric at the pH of electrophoresis can also be used, such as
trialkylammonium propyl sulfonates, where alkyl is methyl, ethyl,
propyl, and longer alkyl chains.
[0084] Another approach involves the generation of a current that
opposes EOF. Typically, this is accomplished by applying a thin
film of metal (e.g., iridium tin oxide or copper) to an external
surface of the capillary. Application of current to the film
generates a relatively small induced current within the capillary
to reverse the EOF (see, e.g., Schasfoort, R. B. M., Schlautmann,
S., Hendrikse, J., and van den Berg, A., "Field-Effect Flow Control
for Microfabricated Fluidic Networks," Science, 286:942-945
(1999)).
[0085] Placing a porous plug at a location upstream from where
sample is introduced (upstream referring to a direction opposite
the flow of proteins through the capillary) can also be utilized to
control EOF. An example illustrating the location of the plug is
illustrated in FIG. 2B where the capillary 100 extends from the
anode reservoir (not shown) at one end and the cathode reservoir at
the other end (not shown). Protein migration is in the direction of
arrow 102 (ie., from the anode to cathode direction). As can be
seen, the porous plug 104 is positioned to be upstream of the
trailing edge 106 of the sample once introduced into the capillary
100. The porous plug 104 is typically formed of a polymeric
material and remains relatively stationary during electrophoretic
runs. Examples of suitable materials from which the plug can be
formed include polymerized acrylamide with diacrylamide
crosslinkers and agarose. Although not intending to be bound by any
particular theory, the porous plug 104 appears to function as a
momentum transfer barrier by blocking replacement of bulk fluid
that in the absence of the plug 104 would move toward the cathode
reservoir.
[0086] In some methods, such as those containing large amounts of
protein and/or a large number of different proteins, EOF should be
reduced to very low levels to allow proteins the opportunity to
focus before the electrophoretic medium begins eluting from the
capillary due to EOF. In certain methods an EOF
of=0.5.times.10.sup.-6 cm.sup.2/V-s (at pH 8.6, and 25 mM
TRIS-phosphate) has been found to allow ample time for the
necessary focusing of proteins before sample elutes from the
capillary. Methods described above can reduce EOFs to these
levels.
[0087] Thus, the foregoing approaches enable fractions to be
collected according to different criteria. Electroelution
techniques, for example, can be used to collect fractions having a
defined pH range. EOF elution and pressure elution, in contrast,
can be used to separate fractions according to time of elution.
Other techniques can also be utilized to elute resolved proteins
after CIEF (see, e.g. Kilar, F., "Isoelectric Focusing in
Capillaries," in CRC Handbook or Capillary Electrophoresis: A
Practical Approach, CRC Press, Inc., chapter 4, pp. 95-109 (1994)).
The controlled elution techniques are useful for enhancing
reproducibility, an important factor in comparative and diagnostic
methods. Such techniques also provide improved tolerance of high
abundance proteins as compared to methods relying on spatial
separation.
[0088] C. Capillarv Zone Electrophoresis (CZE)
[0089] 1. General
[0090] Capillary zone electrophoresis is an electrophoretic method
conducted in free solution without a gel matrix and results in the
separation of molecules such as proteins based upon their intrinsic
charge-to-mass ratio. One advantage to CZE methods is the ability
to run with solvent systems that would normally be incompatible
with typical water soluble gel matrices. Nonaqueous or water
miscible solvent systems can be used to improve the solubility of
hydrophobic and membrane bound proteins that would normally not be
resolved by gel electrophoretic methods. General methods for
conducting the method are described, for example, by McCormick, R.
M. "Capillary Zone Electrophoresis of Peptides," in CRC Handbook of
Capillary Electrophoresis: A Practical Approach, CRC Press Inc.,
chapter 12, pp. 287-323 (1994); Jorgenson, J. W. and Lukacs, K. D.,
J. High Resolut. Chromatogr. Commun., 4:230 (1981); and Jorgenson,
J. W. and Lukacs, K. D., Anal. Chem. 53:1298 (1981)), each of which
is incorporated by reference in its entirety.
[0091] 2. System and Solutions
[0092] In general, the capillaries described above for CIEF are
also suitable for conducting CZE methods. Often the capillaries
have internal diameters of about 50 to 100 microns. Buffer
composition and pH can significantly influence separations since
separations in CZE are based upon charge-to-mass ratios and the
charge of a protein is dependent upon the pH of the surrounding
solution. At the extremes of pH (i.e., below 2 and above 10) it is
typically difficult to achieve resolution of proteins because all
residues are either fully protonated or deprotonated and many
proteins have a similar number of acidic and basic residues per
unit mass. Selectivity is typically enhanced at intermediate pH.
For proteins having a relatively high percentage of acidic
residues, selectivity can often be enhanced near pH 4.5. For those
proteins having a high concentration of basic residues, selectivity
can be enhanced near pH 10.
[0093] In CZE, solutions at the anode and cathode are typically the
same. The buffer utilized can be essentially any buffer, the choice
of buffer being controlled in part by the pH range at which the
electrophoretic method is conducted and its influence on the
detector noise. Examples of useful buffers at low pH include, but
are not limited to, phosphate and citrate; useful buffers at high
pH include Tris/Tricine, borate and CAPS
(3-(cyclohexylamino)-1-propane sulfonic acid). Further guidance
regarding suitable buffers and buffer additives is described by
McCormick, R. M. "Capillary Zone Electrophoresis of Peptides," in
CRC Handbook of Capillary Electrophoresis: A Practical Approach,
CRC Press Inc., chapter 12, pp. 287-323 (1994).
[0094] 3. Elution
[0095] Elution can be accomplished utilizing some of the same
methods described above for CIEF, namely pressure and EOF. As with
CIEF, controlling EOF can be important in certain methods to
prevent electrophoretic medium containing protein from eluting from
the capillary before the proteins within the loaded sample have had
an opportunity to separate. EOF can be controlled using the same
methods utilized for controlling EOF in CIEF methods (e.g., coating
the internal walls of the capillary, using a porous plug and
generating an induced field to counteract EOF). Regulating and
carefully selecting the pH and ionic strength of the
electrophoretic medium is another technique that can be used.
Because EOF results from ionization of the silanol groups on the
interior capillary surface, by conducting CZE at relatively low pH
(e.g., pH 2-5, more typically about pH 3-4) the number of silanol
groups that are ionized is reduced. Such a reduction reduces EOF.
To prevent sample elution prior to complete separation, in certain
analyses the EOF should be reduced to <1.times.10.sup.-4
cm.sup.2/V-s (at pH 8.6 and 25 mM TRIS-phosphate buffer). EOFs of
this level can be obtained using the methods just described.
[0096] Another approach that is described more fully below in the
detection and labeling section is to label proteins in the sample
prior to injecting the sample containing the protein into the
capillary. By selecting labels that preferentially react with
certain functional groups such as amino or carboxyl groups, the
charge-to-mass ratio of certain proteins can be altered. Such
alterations can improve the resolution of proteins during
electrophoresis as well as improve their detectability. (See
Examples 1 and 2 below).
[0097] D. Capillary Gel Electrophoresis (CGE)
[0098] 1. General
[0099] Capillary gel electrophoresis refers to separations of
proteins accomplished by sieving through a gel matrix, resulting in
the separation of proteins by size. In one format, proteins are
denatured with sodium dodecyl sulfate (SDS) so that the
mass-to-charge ratio is determined by this anionic surfactant
rather than the intrinsic mass-to-charge ratio of the protein [50,
2]. This means that proteins can be separated solely on the basis
of size without charge factoring into the degree of separation. The
application of general SDS PAGE electrophoresis methods to
capillary electrophoresis (CGE) is described, for example, by
Hjerten, S., "Free zone electrophoresis," Chromatogr. Rev., 9:122
(1967).
[0100] 2. System and Solutions The type of capillaries and their
size are generally as described above for CZE. A variety of
different buffers can be used, including commercially available
buffers such as the "eCAP SDS" buffers manufactured by Beckman
(see, also, 51, 30, 9 and 5). Various buffer additives can be
utilized to increase resolution. Such additives, include, but are
not limited to, small amounts of organic solvents, such as
N,N-dimethylformamide, cyclohexyldiethylamine,
dimethoxytetraethylene glycol and other polyols (e.g., ethylene
glycol and polyethylene glycol) (see, e.g., [2] and [3]). The use
of such solvents can improve the solubility of proteins in aqueous
solution and enhance protein stability against thermal
denaturation, [52] depress the electroosmotic flow in CZE and CGE
[53], alter the electrical double-layer thickness at the capillary
wall to inhibit protein binding interactions [47] and increase the
viscosity of the running buffer which depresses the electroosmotic
flow. Solvents utilized should be compatible with the polymer
matrix inside the capillary.
[0101] Isotachophoresis (IPE) can be used in certain methods to
increase resolution of proteins. For a general discussion of IPE,
see, for example, B. J. Wanders and Everaerts, F. M.,
"Isotachophoresis in Capillary Electrophoresis," in CRC Handbook of
Capillary Electrophoresis: A Practical Approach, chap. 5, pp.
111-127 (1994), which is incorporated by reference in its entirety.
The velocity of a charged molecule moving through a capillary under
a constant field strength depends on its relative mobility, which
is a function of the mass/charge of the molecule, temperature, and
viscosity of the medium through which it is moving. However, in the
absence of an adequate concentration of highly mobile ions upstream
of the sample ions, all the ions eventually have to migrate at the
speed of the slowest ion once the electric field reaches a
steady-state inside the capillary. This condition causes the anions
to stack in order of their relative mobilities at the interface of
the leading and terminating buffers.
[0102] Under SDS denaturing conditions, all the proteins present in
the sample have nearly identical mass/charges. By using a higher
mass/charge anion in the terminal buffer, one can force the
proteins to move at a constant slow speed through the capillary.
This has two effects. First, proteins "stack" at the terminal edge
of the leading buffer increasing their effective concentration
inside the capillary. Second, any separation between proteins is
based on their size. Therefore, the use of a hybrid IPE-CGE method
in which the IPE is used for sample "stacking" can improve the
resolution possible in the subsequent CGE separation in some
methods.
[0103] Various terminal buffer systems can be utilized in
conjunction with IPE methods. In one system, .epsilon.-aminocaproic
acid (EACA) is used as the terminal electrolyte because it has a
high mass/charge at high pH (>6). Tris(hydroxyethyl)aminomethane
(TRIS) citrate at 0.05M is used as the leading buffer at pH=4.8 and
as an intermediate stacking buffer at pH=6.5. The sample proteins
initially "stack" because EACA has a very low mobility in the pH
6.5 stacking buffer, but once the protein "stack" and EACA reach
the lower pH leading buffer, the mobility of the EACA surpasses
that of the proteins and separation commences (see, e.g., [57]).
This system can be used to create a hybrid single column IPE-CPAGE
system.
[0104] A 2 buffer system for IPE for the separation of proteins
involves dissolving sample in 0.01M acetic acid, which is also used
as the terminal electrolyte. The leading and background buffer was
0.02M triethylamine-acetic acid solution at pH 4.4. The sample in
terminal buffer is sandwiched between the leading and background
buffer. IPE continues until the background buffer overtakes the
leading edge of the terminal buffer, at which point IPE stops and
separation begins (see, e.g., [58]).
[0105] Another IPE approach that can be accomplished with any
running buffer is to dissolve the sample in the running buffer but
diluted to a lower ionic strength. This causes an increase in the
electrical resistance in the capillary where the sample plug is
loaded and correspondingly faster movement of the ions present in
the sample matrix to running buffer boundary. The optimal ionic
strength difference between the sample matrix and the running
buffer is typically about 10-fold (see, e.g., [43]).
[0106] 3. Elution
[0107] In general, the discussion of elution for CZE applies to
CGE. Elution can be accomplished utilizing pressure and EOF. As
with CIEF and CZE, controlling EOF can be important in certain
methods to prevent electrophoretic medium containing protein from
eluting before the proteins within the applied sample have had an
opportunity to separate. The methods described supra for CIEF and
CZE can be used to control EOF at desired levels. To prevent sample
elution prior to complete separation, in certain analyses the EOF
should be reduced to <1.times.10.sup.-4 cm.sup.2/V-s (at pH 8.6
and 25 mM TRIS-phosphate buffer). EOF can be reduced to this range,
for example, by controlling the pH of the buffer, by generation of
a counteracting induced field, capillary coatings and a porous gel
plug.
[0108] E. Labeling and Detection
[0109] As indicated in FIG. 1, electrophoretic solution withdrawn
during the final electrophoretic separation can be directed toward
a detector for the detection and quantitation of protein. This
arrangement provides considerable flexibility with regard to the
nature of detection and does not limit the methods to the standard
gel staining detection techniques common in traditional 2-D gel
electrophoresis analysis. The detector need not be positioned to
detect eluted proteins as shown in FIG. 1, however. In other
arrangements, the detector is adapted so that it can scan resolved
proteins within the separation cavity of the capillary tube itself.
To further enhance detection sensitivity, quantitation and
reproducibility, proteins are labeled at some point prior to
detection in certain methods. Depending upon the particular label
used, signal-to-noise ratios can be achieved which permit the
detection of low abundance proteins.
[0110] Although FIG. 1 depicts a single detector, additional
detectors can be positioned to detect proteins eluting from all or
at least multiple capillaries utilized in the different
electrophoretic methods. One suitable arrangement, for example,
involves utilizing a UV/VIS detector to detect eluting proteins
from early and/or intermediate methods, in part to monitor the
amount of protein being collected within a fraction. Labeling can
then be conducted immediately prior to the final step with
subsequent detection of labeled protein from the final capillary.
If labeling is conducted at some earlier stage, then a detector (or
detectors), can detect labeled protein after all subsequent
electrophoretic methods in the series.
[0111] Proteins can be detected utilizing a variety of methods. One
approach is to detect proteins using a UV/VIS spectrometer to
detect the natural absorbance by proteins at certain wavelengths
(e.g., 214 or 280 nm). In other approaches, proteins in the various
fractions can be covalently labeled through a variety of known
methods with chromagenic, fluorophoric, or radioisotopic labels. A
wide variety of chemical constituents can be used to attach
suitable labels to proteins. Chemistries that react with the
primary amino groups in proteins (including the N-terminus)
include: aryl fluorides [69, 70, 71, 72], sulfonyl chlorides [73],
cyanates [74], isothiocyanates [75], immidoesters [76],
N-hydroxysuccinimidyl esters [77], chlorocarbonates [78],
carbonylazides [78], and aldehydes [79, 80]. Examples of chemical
constituents that preferentially react with the carboxyl groups of
proteins are benzyl halides [78, 81, 82] and carbodiimide [83],
particularly if stabilized using N-hydroxysuccinimide [84]. Both of
these carboxyl labeling approaches are expected to label carboxyl
containing amino acid residues (e.g., aspartate and glutamate)
along with that of the C-terminus. In addition, tyrosine residues
can be selectively [.sup.135I]-iodinated to allow radiochemical
detection.
[0112] As alluded to supra, labeling can be performed at different
points prior to detection. In general, however, the decision when
to label proteins during the overall analysis depends on the
particular CE methods utilized in the series. For example, if CIEF
is utilized in one of the dimensions, then proteins are typically
labeled after CIEF. Labeling can alter the pI of the proteins,
thereby changing the locations at which the proteins focus during
CIEF. This is not inherently problematic, but it means that the
results cannot be compared with typical archival 2-D gel
electrophoresis results that include an IEF dimension. Thus, to
allow comparison with results obtained using more traditional
approaches, it can be advantageous to delay labeling until after
CIEF. Certain labels, however, can be utilized that have a minimal
effect on the IEF pattern (see, e.g., U.S. Pat. No. 5,320,727, and
Jackson, P. et al., Electrophoresis, 9:330-339 (1998), both of
which are incorporated by reference in their entirety).
[0113] If the decision is made not to label protein prior to
conducting CIEF for the foregoing reasons, proteins eluting from
the CIEF capillary or proteins in collected fractions can
nonetheless be detected by detecting UV absorbance or intrinsic
fluorescence of the aromatic amino acids (e.g., tryptophan,
phenylalanine and tyrosine). If, however, proteins are labeled
prior to CIEF, any effect of pH on the absorptivity of the dye or
fluorophor label can be mitigated by spin dialysis and buffer
exchange to a constant pH before measurement, or by resuspending a
constant collected volume into a higher ionic strength buffer of a
constant pH.
[0114] Whereas labeling is generally conducted after the CIEF
dimension to avoid altering isoelectric focusing patterns, there
can be advantages to labeling proteins prior to a CZE dimension.
Depending upon the composition of the protein-containing sample,
labeling of proteins can affect the charge-to-mass ratio of the
labeled proteins. For protein mixtures wherein the proteins have
similar charge-to-mass ratios, the use of labels that
preferentially react with particular residues can alter the
charge-to-mass ratios sufficiently such that enhanced resolution is
achieved. For example, a group of proteins can initially have a
similar charge-to-mass ratio. However, if the proteins within the
group are labeled with a neutral label that reacts primarily with
lysine groups, proteins having a high number of lysine groups will
bear more label and have a greater alteration in the charge-to-mass
ratio than proteins having a lower number of lysine residues. This
differential effect can translate into enhanced fractionation
during electrophoresis. (See Examples 1 and 2).
[0115] A variety of labels that preferentially react with specific
residues are available for use. The reactive functionality on the
label is selected to ensure labeling of most or all of the
components of interest. For example, sulfophenylisothiocyanate can
be used to selectively label lysine residues [20], altering their
charge from positive (below a pH of 10) to negative (above a pH of
0.5). Similarly, phenylisothiocyanate can be used to neutralize the
lysine and N-terminal positive charges at all pH. Dansyl chloride
can be used to lower the pH at which lysine and N-terminal residues
carry a net positive charge. The addition of amino functional alkyl
ammonium salts to aspartic and glutamic acid residues, such as
through carbodiimide coupling, alters their charge from negative to
positive at low pH.
[0116] There is somewhat greater flexibility in the time at which
proteins are labeled relative to CGE. In some instances,
pre-labeling is advantageous in that the separation can be viewed
as it occurs and in that detection can be performed at the end
without further labeling. With pre-labeling there are also fewer
fractions of proteins to label. Pre-labeling methods for ID-PAGE
are described, for example, by Hames, B. D. in Gel Electrophoresis
of Proteins: A Practical Approach, (Hames, B. D. and Rickwood, P.,
Eds.) 2.sup.nd ed., pp. 67-68, Oxford University Press, Oxford
(1990) and Rose, D. R. J. and J. W. Jorgensen, "Post-capillary
fluorescence detection in capillary zone electrophoresis using
o-phthaldialdehyde," J. Chromatogr., 447:117 (1988), which are
incorporated by reference in their entirety.
[0117] Although certain types of labels are preferred to enhance
separation in CZE methods, in general the label utilized during
labeling in one of the methods of the invention can be quite
diverse. In general the label should not interfere with
fractionation during electrophoresis and should emit a strong
signal so that even low abundance proteins can be detected. The
label preferably also permits facile attachment to proteins.
Suitable labels include, for example, radiolabels, chromophores,
fluorophores, electron dense agents, NMR spin labels, a chemical
tag suitable for detection in a mass spectrometer, or agents
detectable by infrared spectroscopy or NMR spectroscopy for
example. Radiolabels, particularly for spacially resolved proteins,
can be detected using phosphor imagers and photochemical
techniques.
[0118] Certain methods utilize fluorophores since various
commercial detectors for detecting fluorescence from labeled
proteins are available. A variety of fluorescent molecules can be
used as labels including, for example, fluorescein and fluorescein
derivatives, rhodamine and rhodamine derivatives, naphythylamine
and naphthylamine derivatives, benzamidizoles, ethidiums,
propidiums, anthracyclines, mithramycins, acridines, actinomycins,
merocyanines, coumarins, pyrenes, chrysenes, stilbenes,
anthracenes, naphthalenes, salicyclic acids, benz-2-oxa-1-diazoles
(also called benzofurazans), fluorescamines and bodipy dyes.
Specific examples of suitable fluorescent labels are listed in
Table 1 below. A variety of appropriate fluorescent dyes are
commerciaily available from Sigma Chemical Co. (St. Louis, Mo.) and
Molecular Probes, Inc. (Eugene, Oreg.).
1TABLE 1 Labels and Labeling Methods Linkage Label Source Formed
Amine Labeling 2,4,6-trinitrobenzenesulfonic acid Aldrich Aryl
amine Lissamine .TM. rhodamine B sulfonyl Molecular Sulfonamide
chloride Probes 2',7'-dichlorofluoroscein-5- Molecular Thiourea
isothiocyanate Probes 4,4-difluoro-5,7-dimethyl-4-bora- Molecular
Amide 3a,4a-diaza-s-indacene-3-propionic Probes acid,
sulfosuccinimidyl ester Nahthalene-2,3-dicarboxylaldehyde Molecular
Isoindole Probes Carboxyl Labeling 5-(bromomethyl)fluorescein
Molecular Ester Probes N-cyclohexyl-N'-(4-(dimethylamino) Molecular
N-Acylurea naphthyl)carbodiimide Probes
1-ethyl-3-(3-dimethylaminopropyl)- Pierce Amide carbodiimide
hydrochloride with N- Aldrich hydroxysuccinimide and 5- Molecular
aminofluorescein Probes
[0119] In some instances, the proteins separated by the methods of
the invention are subjected to further analysis by mass
spectroscopy. In such instances, particular labels can be utilized
to enhance separation of mass fragments into certain parts of the
mass spectrum. Suitable labels in such methods are set forth more
fully in copending application Ser. No. ______, entitled "Methods
for Protein Sequencing," having attorney docket number
020444-000300US, and filed on the same date as the current
application. This application is incorporated herein by reference
in its entirety.
[0120] Quantitation of detected signals can be performed according
to established methods. Peak height and peak area are typically
used to quantify the amount of each resolved protein in the final
electrophoretic dimension. In some methods, the peak height, peak
width at the half height, peak area, and elution time for each peak
are recorded. Peak shape (determined as the height to width ratio)
can be used as a measure of the quality of the separation method.
The resolution potential of the method can be determined by
correlating the MW of the protein with the elution time (see, e.g.,
[30] and [11]). By dividing the overall run time by the average
peak width of each protein an estimate of the total number of
proteins that can be resolved by the method (e.g., proteins
separated by at least one peak width can be considered a "resolved"
protein) can be obtained. The reproducibility of the MW estimate
can be determined by two methods. In one method, the apparent MW
determined for each protein in three replicate runs by establishing
the standard curve from one run and using that curve to determine
the MW based on elution time from each subsequent run are compared
(see, e.g., [21]). In the second approach, the overall error of the
method is determined from the standard deviation in the slope of
the standard curve created using the data from all three replicate
runs.
[0121] The labeling and direct detection approaches that can be
used with certain methods of the invention can yield improved
reproducibility in the quantification of relative protein
expression levels compared to the staining and imaging methods
utilized in conventional 2-D gels. Staining techniques frequently
yield poorly quantitative results because varying amounts of stain
are incorporated into each protein and the stained protein must be
detected and resolved against the stained background of the gel or
electroblotting substrate. Moreover, since the methods utilize
combinations of electrophoretic methods, an electropherogram that
is directly comparable to archived 2-D gel image data is still
obtained. This means that the methods remain comparable to 2-D gel
information as compared to other non-electrophoretic based
separations (e.g., LC/MS/MS).
[0122] F. Exemplary Systems
[0123] The methods of the invention are amenable to a variety of
different electrophoretic methods. The controlled elution
techniques whereby defined fractions are separated spatially,
physically or by time, and the labeling and detection methods can
be utilized in a number of different electrophoretic techniques. As
noted above, the number of electrophoretic methods linked in series
is at least two, but can include multiple additional
electrophoretic methods as well. In some instances, each
electrophoretic method in the series is different; whereas, in
other instances certain electrophoretic methods are repeated at
different pH or separation matrix conditions.
[0124] Despite the general applicability of the methods, as noted
above CIEF, CZE and CGE methods are specific examples of the type
of electrophoretic methods that can be utilized according to the
methods of the invention. In certain methods, only two methods are
performed. Examples of such methods include a method in which CIEF
is performed first followed by CGE. Labeling is typically performed
after CIEF with detection subsequent to elution of protein from the
CGE capillary. Protein eluting from the CIEF capillary can be
detected using a UVVIS spectrometer at 214 or 280 nm, for example.
In another system, the first method is CZE and the final method is
CGE. With this arrangement, labeling is typically performed prior
to CZE to enhance resolution as described supra. Detection
generally is not performed until the completion of the final
electrophoretic separation. A third useful approach involves
initially conducting CIEF followed by CZE and CGE. Labeling for
such a system is typically done after CIEF and before CZE. Labeling
at this point in the overall method avoids alteration of CIEF
patterns (see supra) and allows for greater resolution during CZE.
Detection is generally conducted at the conclusion of CGE (i.e.,
with resolved protein within the capillary or after the proteins
have eluted from the capillary). These are specific examples of
systems that can be utilized; it should be understood that the
invention is not limited to these particular systems. Other
configurations and systems can be developed using the techniques
and approaches described herein.
[0125] IV. Samples
[0126] The methods of the invention can be used with a wide range
of sample types. Essentially any protein-containing sample can be
utilized with the methods described herein. The samples can contain
a relatively small number of proteins or can contain a large number
of proteins, such as all the proteins expressed within a cell or
tissue sample, for example.
[0127] Samples can be obtained from any organism or can be mixtures
of synthetically prepared proteins or combinations thereof. Thus,
suitable samples can be obtained, for example, from microorganisms
(e.g., viruses, bacteria and fungi), animals (e.g., cows, pigs,
horses, sheep, dogs and cats), hominoids (e.g., humans,
chimpanzees, and monkeys) and plants. The term "subject" as used to
define the source of a sample includes all of the foregoing
sources, for example. The term "patient" refers to both human and
veterinary subjects. The samples can come from tissues or tissue
homogenates or fluids of an organism and cells or cell cultures.
Thus, for example, samples can be obtained from whole blood, serum,
semen, saliva, tears, urine, fecal material, sweat, buccal, skin,
spinal fluid, tissue biopsy or necropsy and hair. Samples can also
be derived from ex vivo cell cultures, including the growth medium,
recombinant cells and cell components. In comparative studies to
identify potential drug or drug targets (see infra), one sample can
be obtained from diseased cells and another sample from
non-diseased cells, for example.
[0128] Sample preparation for the different electrophoretic
techniques is set forth above. If the sample contains cellular
debris or other non-protein material that might interfere with
separation during electrophoresis, such materials can be removed
using any of a variety of known separation techniques including,
for example, forcibly exuding the sample through sieve material,
filtration and centrifugation. Samples whose ionic strength is
particularly high can be desalted using established techniques such
as dialysis and dilution and reconcentration.
[0129] In some instances in which the sample contains salts or
other interfering components, buffer exchange can be performed to
improve IPE "stacking" and improve reproducibility in elution times
and peak shapes for electrophoretic methods. One useful way to
implement dialysis to remove interfering compounds is to collect
fractions directly in the dialysis chamber of a spin dialysis tube
(Gilson/Amicon). The sample can then be spin dialyzed and
resuspended in a 10-fold dilution of the running buffer to be
utilized in the next electrophoretic separation of the series. This
procedure has the advantages that:
[0130] (1) in the case of CIEF, larger volumes of buffers can be
used during electroelution of each fraction without diluting the
proteins in each fraction, (2) the same sample volume can be used
for each fraction injected into the second dimension and (3)
smaller more concentrated sample volumes can be used in the second
dimension because the dialyzed proteins can be resuspended in
almost any buffer volume after dialysis.
[0131] V. Variations
[0132] A. Further Analysis
[0133] The methods of the invention need not end with the last
electrophoretic method of the series. As illustrated in FIG. 1,
resolved proteins can be further analyzed by non-electrophoretic
methods. Examples of such methods include infra-red spectroscopy,
nuclear magnetic resonance spectroscopy, UV/VIS spectroscopy and
complete or partial sequencing. Coupling the current
electrophoretic-based method to various mass spectroscopy (MS)
methods is one specific example of further analysis that can be
conducted. A variety of mass spectral techniques can be utilized
including several MS/MS methods and Electrospray-Time of Flight MS
methods (see, e.g., [61], [62], [63], and [64]). Such methods can
be used to determine at least a partial sequence for proteins
resolved by the electrophoretic methods such as a protein sequence
tag (for a discussion or protein sequence tags, see, e.g., [65] and
[66]). Further discussion regarding combining the electrophoretic
separations described herein with mass spectral analysis is set
forth in U.S. provisional application 60/130,238 entitled "Rapid
and Quantitative Protein Expression and Sequence Determination,"
filed Apr. 20, 1999, and to which this application claims benefit
and which is incorporated by reference in its entirety. Other mass
spectral methods that can be combined with the methods of the
present invention are described in copending U.S. application Ser.
No. ______, entitled "Methods for Protein Sequencing," and having
attorney docket number 020444-000300US, and copending U.S.
application Ser. No. ______, entitled "Polypeptide Fingerprinting
Methods and Bioinformatics Database System, and having attorney
docket number 020444-000100US, both filed on the same date as the
current application and both being incorporated by reference in
their entirety.
[0134] B. Microfluidic Systems
[0135] 1. Examples of Configurations
[0136] In another variation, the capillaries are part of or formed
within a substrate to form a part of a microfluidic device that can
be used to conduct the analyses of the invention on a very small
scale and with the need for only minimal quantities of sample. In
these methods, physical fractions of samples typically are not
collected. Instead, resolved proteins are separated spatially or by
time. Methods for fabricating and moving samples within
microfluidic channels or capillaries and a variety of different
designs have been discussed including, for example, U.S. Pat. Nos.
5,858,188; 5,935,401; 6,007,690; 5,876,675; 6,001,231; and
5,976,336, all of which are incorporated by reference in their
entirety.
[0137] An example of a general system 150 that can be used with the
methods of the present invention is depicted in FIG. 3A. The
capillaries or channels are typically formed or etched into a
planar support or substrate. A separation capillary 152 extends
from an anode reservoir 154 containing anolyte to a cathode
reservoir 156. The anode reservoir 154 and the cathode reservoir
156 are in electrical contact with an anode and cathode 158, 160,
respectively. A sample injection channel 162 runs generally
perpendicular to the separation capillary 152 and one end
intersects at an injection site 164 slightly downstream of the
anode reservoir 154. The other end of the sample injection
capillary 162 terminates at a sample reservoir 166, which is in
electrical communication with a sample reservoir electrode 168. A
detector 170 is positioned to be in fluid communication with
electrophoretic medium passing through the separation capillary 152
and is positioned downstream of the sample injection site 164 and
typically somewhat upstream of the cathode reservoir 156. In this
particular configuration, fractions are withdrawn into the cathode
reservoir 156. Movement of electrophoretic medium through the
various channels is controlled by selectively applying a field via
one or more of the electrodes 158, 160 168. Application of a field
to the electrodes controls the magnitude of the EOF within the
various capillaries and hence flow through them.
[0138] An example of another configuration is illustrated in FIG.
3B. This system 180 includes the elements described in the system
shown in FIG. 3A. However, in this arrangement, spacially or
temporally resolved fractions can be withdrawn at multiple
different locations along the separation capillary 152 via exit
capillaries 172a, 172b and 172c. Each of these capillaries includes
a buffer reservoir 176a, 176b, 176c, respectively, and is in
electrical communication with electrodes 174a, 174b, 174c,
respectively. Movement of electrophoretic medium along separation
capillary 152 and withdrawal of fractions therefrom into the exit
capillaries 172a, 172b and 172c can be controlled by controlling
which electrodes along the separation capillary 152 and which of
the exit capillary electrodes are activated. Alternatively, or in
addition, various microfluidic valves can be positioned at the exit
capillaries 172a, 172b and 172c to control flow. Typically,
additional detectors are positioned at the various exit capillaries
172a, 172b and 172c to detect protein in fractions withdrawn into
these capillaries.
[0139] The configuration illustrated in FIG. 3B can be used in a
number of different applications. One example of an application for
which this type of system is appropriate is a situation in which
the type of samples being examined have been well characterized. If
for example, certain fractions of proteins of interest have been
previously established to fractionate at a particular location in
the separation capillary 152, then the exit capillaries 172a, 172b
and 172c can be positioned at those locations to allow for
selective removal of the protein fraction(s) of interest.
[0140] In still another configuration, multiple exit capillaries
branch from the end of the separation capillary 152 near the
cathode reservoir 156, each exit capillary for withdrawing and
transporting separate fractions. In this configuration also,
withdrawal of fractionated protein from the separation capillary
can be controlled by regulating EOF within the various capillaries
and/or by microfluidic valves.
[0141] Other components necessary for conducting an electrophoretic
analysis can be etched into the support, including for example the
reservoirs, detectors and valves discussed supra.
[0142] 2. Substrates
[0143] The substrate upon which the capillary or micro-channel
network of the analytical devices of the present invention are
formed can be fabricated from a wide variety of materials,
including silicon, glass, fused silica, crystalline quartz, fused
quartz and various plastics, and the like. Other components of the
device (e.g., detectors and microfluidic valves) can be fabricated
from the same or different materials, depending on the particular
use of the device, economic concerns, solvent compatibility,
optical clarity, mechanical strength and other structural concerns.
Generally, the substrate is manufactured of a non-conductive
material to allow relatively high electric fields to be applied to
electrokinetically transport the samples through the various
channels.
[0144] In the case of polymeric substrates such as plastics, the
substrate materials can be rigid, semi-rigid, or non-rigid, opaque,
semi-opaque or transparent, depending upon the use for which the
material is intended. Plastics which have low surface charge when
subjected to the electric fields of the present invention and thus
which are of particular utility include, for example,
polymethylmethacrylate, polycarbonate, polyethylene terepthalate,
polystyrene or styrene copolymers, polydimethylsiloxanes,
polyurethane, polyvinylchloride, polysulfone, and the like.
[0145] Devices which include an optical or visual detector are
generally fabricated, at least in part, from transparent materials
to facilitate detection of components within the separation channel
by the detector.
[0146] 2. Channel Structure/Formation
[0147] The size and shape of the channels or capillaries formed in
the substrate of the present devices can have essentially any
shape, including, but not limited to, semi-circular, cylindrical,
rectangular and trapezoidal. The depth of the channels can vary,
but tends to be approximately 10 to 100 microns, and most typically
is about 50 microns. The channels tend to be 20 to 200 microns
wide.
[0148] Manufacturing of the channels and other elements formed in
the surface of the substrate can be carried out by any number of
microfabricating techniques that are known in the art. For example,
lithographic techniques may be employed in fabricating glass or
quartz substrates, for example, using established methods in the
semiconductor manufacturing industries. Photolithographic masking,
plasma or wet etching and other semiconductor processing
technologies can be utilized to create microscale elements in and
on substrate surfaces. Alternatively, micromachining methods, such
as laser drilling, micromilling and the like, can be utilized.
Manufacturing techniques for preparing channels and other elements
in plastic have also been established. These techniques include
injection molding techniques, stamp molding methods, using for
example, rolling stamps to produce large sheets of microscale
substrates, or polymer microcasting techniques, wherein the
substrate is polymerized within a micromachined mold.
[0149] Further guidance regarding other designs and methods for
using such microfluidic devices such as described above can be
found, for example, in U.S. Pat. Nos. 5,858,188; 5,935,401;
6,007,690; 5,876,675; 6,001,231; and 5,976,336, all of which are
incorporated by reference in their entirety.
[0150] C. Preliminary Separation by Non-Electrophoretic
Technique
[0151] The methods can also include an initial separation by a
non-electrophoretic technique prior to commencing the
electrophoretic separations. Essentially any type of technique
capable of separating proteins can be utilized. Suitable methods
include, but are not limited to, fractionation in a sulfate
gradient, HPLC, ion exchange chromatography and affinity
chromatography.
[0152] VI. Exemplary Utilities
[0153] The methods and apparatus of the invention can be utilized
to detect, characterize and/or identify many proteins (e.g.,
hundreds or thousands of proteins in some methods) by controlling
elution of fractionated proteins and utilizing various labeling and
detection techniques. Consequently, the methods have multiple
utilities including, but not limited to, various analytical
applications (e.g., monitoring certain protein levels as a function
of external stimuli, or detecting specific proteins in complex
compositions for identification purposes), clinical applications
(e.g., detecting and/or monitoring compositions of normal and
diseased cells and tissues, diagnosing or monitoring disease,
testing drug candidates for therapeutic efficacy and toxicity
testing) and molecular biology and genetic research (e.g.,
characterizing or monitoring molecular expression levels of gene
products and determining the effects of the addition, mutation,
deletion or truncation of a particular gene). In general, the
methods and apparatus have utility in proteome research.
[0154] More specifically, the invention can be used in the
development of protein databases in which, for example, proteins
expressed under particular conditions are isolated, quantified, and
identified. Using the controlled elution and detection methods
described herein, certain methods can be utilized to determine and
catalog a variety of chemical and physical characteristics of the
resolved proteins, including, but not limited to, pI, and/or
apparent molecular weight and/or relative abundance of proteins
within a sample. This information can be further cross referenced
with a variety of information regarding the source of the sample
and the method by which it was collected. Examples of such
information include genus, species, age, race, sex, environmental
exposure conditions, subject's health, tissue type, method of
sample collection and method of sample preparation prior to
electrophoresis.
[0155] The methods also have value in a variety of comparative
studies that can be utilized to identify potential drug targets
and/or candidates. For example, the methods can be utilized to
identify proteins that are differentially expressed in diseased
cells as compared to normal cells. Such differentially expressed
proteins can serve as targets for drugs or serve as a potential
therapeutic. In a related fashion, the methods can be used in
toxicology studies to identify proteins that are differentially
expressed in response to particular toxicants. Such differentially
expressed proteins can serve as potential targets or as potential
antidotes for particular toxic compounds or challenges. The
detection and labeling techniques of the invention can facilitate
such investigations because these techniques enable even low
abundance proteins to be detected and because enhanced
reproducibility makes it easier to identify real differences in
expression between different samples.
[0156] Proteornic studies using certain methods of the invention
can detect mutations that result in premature termination of the
gene transcript or in amino acid substitutions in the resulting
gene product. The methods can also detect post translational
modification events associated with disease that are not readily
detectable or possible to detect using functional genomics. For
example, proteomic methods can detect differences in protein
folding, glycosylation patterns, phosphorylation events, and
degradation rates.
[0157] The results of comparative studies are transferable to a
variety of diagnostic applications. For example, the "marker" or
"fingerprint" proteins identified during comparative studies as
being characteristic of a particular disease can be used to
diagnosis individuals to determine if they have the disease
correlated with the marker. These markers can also be used in
medical screening tests. Once such protems have been identified, it
is not necessary to examine all fractions. Instead, only those
fractions potentially containing the marker proteins need be
examined. The reproducibility of the methods facilitates such
analyses. For systems integrated onto a chip or support (see
supra), capillaries can be positioned at the appropriate locations
along the separation cavity to withdraw only the relevant fractions
potentially containing the marker protein(s) of interest.
[0158] As an example of a diagnostic application, proteomic
analysis can be utilized in identifying diagnostic markers (e.g.,
cell surface antigens or serum proteins) for immunodiagnostic
assays. Purified samples of putative diagnostic proteins are
recovered during proteomic analysis, and can be used to generate
antibodies having specific binding affinity to the proteins. Such
antibodies can be used to understand the link between the marker
protein and the disease through immunological staining to localize
the protein in diseased cells or to rapidly screen patients for the
presence of the protein, showing its statistical link to the
disease.
[0159] The methods of the invention have further utility in
conducting structure activity studies. For instance, the methods
can be used to determine the effect that certain chemical agents or
combination of agents have on protein expression patterns.
Alterations to the agent or combination can then be made and
protein expression reassessed to determine what effect if any the
alteration has on protein expression. Such studies can be useful,
for example, in making derivatives of a lead compound identified
during initial drug screening trials.
[0160] Metabolic engineering studies can also be conducted using
the methods of the invention. In such studies, a gene can be
genetically engineered to include certain changes or the promoter
of a gene be genetically engineered to increase or decrease its
relative expression level. The methods described herein can then be
used to determine what effect, if any, the genetically engineered
changes have on proteins other than the protein encoded by the
genetically engineered gene.
[0161] The following examples are offered to illustrate, but no to
limit the claimed invention.
EXAMPLE 1
CZE Separation of Unlabeled Proteins
[0162] Each of five proteins (see Table 2) were obtained from
Sigma-Aldrich and were suspended at 5 mg/ml in an aqueous
denaturing sample buffer consisting of 25 mM
tris(hydroxymethyl)aminomethane phosphate (pH 4.0), 0.5% by weight
IGEPAL CA-630 (obtained from Sigma-Aldrich, Cat # 13021), and 1% by
weight tris(2-carboxyethylphosphin- e)hydrochloride (TCEP, obtained
from Pierce, Cat # 20490ZZ). The protein samples were denatured in
this sample buffer by heating at 95.degree. C. for 15 min. Each of
the five denatured protein samples were diluted into a cZE sample
buffer to create a final solution consisting of 25 mM
tris(hydroxymethyl)aminomethane phosphate buffer (pH 4.0), 8 M
Urea, and a final concentration of 0.2 mg/ml of each of the five
proteins. Control samples were also prepared of each denatured
protein separately at 0.5 mg/ml final concentration in the same
sample buffer.
2TABLE 2 Protein Standards Protein Cat # pl MW (kDa) Hen egg white
conalbumin C 0755 6.0, 6.3, 6.6 76.0 Bovine serum albumin B 4287
5.4, 5.5, 5.6 66.2 Carbonic anhydrase II T 6522 4.5 21.5 Rabbit
muscle GAPDH G 2267 8.3, 8.5 36.0 Bovine ribonuclease A R 5503 9.6
13.7
[0163] The mixed protein sample and each of the control samples
were run by CZE in a 60 cm.times.75 .mu.m fused silica capillary
(Beckman Coulter). An 800 .mu.m detection window was located 50 cm
from the anodic end of the capillary. A 160 nl sample volume was
pressur injected at the anodic end and the separations conducted at
500 V/cm in a 25 mM TRIS-phosphate and 8 M urea running buffer at
pH 4.0. Protein detection was accomplished by UV adsorption at 214
nm.
[0164] The individual unlabeled proteins were not resolved under
these conditions (see FIG. 4). The electrophoretic mobility of each
protein was determined from replicate runs of the individual
protein controls (FIG. 5) and correlated with the predicted mass to
charge ratio of the proteins at pH 4.0 (FIG. 6). The mass to charge
ratio for each of the unlabeled proteins was determined from the
published protein sequences obtained through Genbank in the manner
described by Canter, C. R. and Schimmel, P. R., Biophysical
Chemistry, W.H. Freeman and Co., New York, (1980), which is
incorporated by reference in its entirety.
EXAMPLE 2
CZE Separation of Labeled Proteins, with Fraction Collection
[0165] Each of the five proteins described in Example 1 was
suspended at 10 mg/ml in a denaturing buffer containing 1% by
weight of sodium dodecyl sulfate and 1% by volume
2-mercaptoethanol. The proteins were denatured in this buffer by
heating at 95.degree. C. for 15 min. The denatured protein samples
were labeled with 4-sulfophenylisothiocyanate (SPITC) obtained from
Sigma-Aldrich (Cat # 85,782-3) and used as supplied. Labeling was
accomplished by adding 0.01 ml of triethylamine, 0.01 ml of 2 M
acetic acid and 0.02 ml of a 10% by weight solution of SPITC in
water to 0.1 ml of each denatured protein sample. The reaction
mixture was heated at 50.degree. C. for 24 h.
[0166] A quantity of 0.05 ml of each of the SPITC-labeled protein
standards was mixed together and separated by cZE as described in
Example 1, with the exception that the pH of the separation buffer
was adjusted to 3.0. The individual SPITC-labeled proteins were
resolved (FIG. 7). Thus, this example taken in view of the results
for Example 1 in which unlabeled proteins were poorly resolved
demonstrates the positive effect that labeling can have when done
prior to a cZE separation. Fractions were collected by
electroelution into separate vials containing the separation buffer
at the times indicated. The identities of the SPITC-labeled
proteins were determined by subsequent cGE analysis of the
fractions.
EXAMPLE 3
CIEF First Dimension Separation with Fraction Collection
[0167] Bovine Serum Albumin, Carbonic Anhydrase, and Conalbumin
were used as supplied from Sigma-Aldrich (Table 2). Each protein
was denatured as described in Example 1. A 0.01 ml aliquot of each
denatured protein sample was added to 0.2 ml of the CIEF focusing
buffer. The CIEF focusing buffer consisted of 0.4% by weight
hydroxymethyl cellulose solution (Beckman-Coulter eCAP CIEF Gel
Buffer, Cat # 477497) containing 1% by volume pH 3-10 Ampholytes
(Fluka, Cat # 10043) and 1% by weight 3-[(3-cholamidopropyl)
dimethylammonio]-1-propane sulfonate.
[0168] A poly(ethylene glycol)-coated 60 cm long 0.1 mm internal
diameter fused silica capillary (Supelcowax 10, Supelco, Cat #
25025-U) was filled with the protein sample in the focusing buffer.
The capillary contents were focused between 10 mM phosphoric acid
and 20 mM NaOH reservoirs for 7.5 min at 500 V/cm and 25 C. A 0.5
psi pressure gradient was then applied between the anolyte and
catholyte reservoirs to facilitate the elution of the focused
proteins in the direction of the electroosmotic flow.
[0169] The protein peaks were detected by monitoring the
ultraviolet absorption at 214 nm through an optical window in the
capillary positioned 50 cm from the low pH end. The current through
the capillary was also monitored (FIG. 8). Fractions (B-G) were
collected into 0.05 ml of 20 mM NaOH contained in separate
reservoir vials for the times depicted (FIG. 8). Only fractions F
and G were found to contain protein (see Example 4). Fraction G was
found to contain carbonic anhydrase and no conalbumin or bovine
serum albumin. Conalbumin and bovine serum albumin were found to
coelute in the peak observed in fraction F. This experiment
illustrates the partial separation of a mixture of proteins in a
single dimension. Further resolution was achieved in the second
dimension (see Example 4).
EXAMPLE 4
CGE Second Dimension Separation of CIEF Fractions
[0170] Each of the CIEF fractions (B-G) collected during the CIEF
separation described in Example 3 were evaporated in a Savant Model
SC210A Spin-Vap to a final volume of 0.005 ml to concentrate any
protein present in the fraction. A quantity 0.01 ml of SDS sample
buffer was added to each protein concentrate. The SDS sample buffer
consisted of 0.1 ml of eCAP SDS sample buffer (Beckman Coulter, Cat
# 241525), 0.01 ml of eCAP Orange G Reference Marker (Beckman
Coulter, Cat # 241524), and 0.09 ml of anhydrous glycerol.
[0171] Each sample was then run in CGE mode using a linear
poly(acrylamide)-coated fused silica capillary 60 cm long with a
100 .mu.m internal diameter. The eCAP SDS 14-200 Gel buffer
(Beckman-Coulter Cat# 477416) was used for the separation and in
both reservoirs. The separation was conducted at 20.degree. C. and
500 V/cm for 50 min. Ultraviolet detection of the proteins was
accomplished at 214 nm through an optical window positioned 50 cm
from the sample injection end of the capillary. Molecular weight
calibration was conducted in a separate run using eCAP MW Standards
(Beckman-Coulter Cat # 477418) as described by the manufacturer. A
100 sec sample injection at 0.5 psi was used to load each sample
into the capillary.
[0172] The resulting electropherograms showed no detectable protein
in any cIEF fraction except fractions F (FIG. 9) and G (FIG. 10).
The molecular weight of the two proteins seen in fraction F (FIG.
9) correspond to that of bovine serum albumin and conalbumin (Table
2). The molecular weight of the protein seen in fraction G (FIG.
10) corresponded to that of carbonic anhydrase (Table 1). It is
observed that the second cGE dimension was necessary to fully
resolve bovine serum albumin from conalbumin, which were not
resolved by a single cIEF mode (Example 3).
EXAMPLE 5
Use of Methods in Proteomics Analysis for Distinguishing Between
Healthy and Cancerous Tissue
[0173] This example illustrates the use of the present invention
for distinguishing between healthy and cancerous tissue. The
present invention can be used to directly analyze the protein
expression pattern of healthy and cancerous and metastasized
tissues to elucidate patterns of gene expression and translate such
relations to the various aspects of onset, staging and metastases
in cancers, such as prostrate, breast, colon and skin.
[0174] The methods of the invention can significantly decrease the
time necessary to conduct functional genomics analysis of the
mechanism of disease and can lead to the identification of new
therapeutic targets, diagnostic markers, and drug products (i.e.,
where a specific cellular protein may itself act as a therapeutic
agent). By using proteomic analysis the number of genes that must
be investigated is reduced 10-fold (from the 50,000 to 150,000
human genes to the 2,000-10,000 genes actually being expressed to
form proteins in the target tissue). Through quantitative
comparison of the protein expression pattern of healthy and
diseased tissue, the number of candidate genes that may play roles
in the progression of the disease is further reduced about
100-fold. Finally, through the subsequent generation of protein
sequence tags (PTSs; i.e., a partial amino acid sequence) each of
the proteins that show differential expression can be uniquely
identified in a manner that allows them to be tracked back to the
genome for complete sequencing (e.g., mutation detection).
[0175] Initially, tissue samples are obtained from diseased
subjects and control subjects (e.g., individuals not known to have
the particular cancer being studied). The tissue samples from each
individual are homogenized according to known methods. Depending
upon the sample, the resulting homogenate is filtered or
centrifuged to remove cellular debris. Samples are taken from the
homogenate and the proteins therein denatured by adjusting the
samples to contain urea (6-8 M), detergent (e.g., 1% by weight
sodium dodecyl sulfate) and 1% by weight dithiothreitol. Samples
are heated at 95.degree. C. for 15 minutes to speed
denaturation.
[0176] Samples (5 .mu.l) are then electrophoresed by CIEF on a
column (75 micron inside diameter by 60 cm long). Anolyte is
initially 10 mM phosphoric acid and the catholyte is initially 20
mM sodium hydroxide. Separations are conducted at 500 V/cm.
Fractions of resolved proteins are eluted by increasing the sodium
chloride concentration of the catholyte solution from 10 mM to 100
mM in 96 incremental units. Fractions are collected by sequentially
inserting the high pH end of the capillary into 200_l of each salt
concentration in catholyte solution contained in the wells of a 96
well plate. The separation current is allowed to reequilibrate
before the capillary end is moved to the next fraction.
[0177] Prior to labeling, fractions are concentrated using a rotary
evaporator. Protein in the collected fractions is labeled by
reacting the proteins with fluoroscein isothiocyanate as described
in Example 2 for sulfophenylisothiocyanate.
[0178] Fractions containing the labeled proteins are separately
electrophoresed by CZE. The labeled proteins are diluted into a CZE
sample buffer to form a final solution consisting of 25 mM
tris(hydroxymethyl)aminomethane phosphate buffer (pH 4.0), 8 M
urea, and a final concentration of about 1 mg/ml of protein. The
mixed protein sample and each of the control samples are run in CZE
mode in a 60 cm.times.75 .mu.m fused silica capillary (Beckman
Coulter). An 800 .mu.m window is located 50 cm from the anodic end
of the capillary. A 160 nl sample volume is pressure injected at
the anodic end and the separations conducted at 500 V/cm in a 25 mM
TRIS-phosphate and 8 M urea running buffer at pH 4.0. Proteins are
eluted by the residual EOF in the capillary. Fractions are again
collected on the basis of elution time in the wells of a 96 well
microtiter plate as the capillary is progressively advanced from
one well to the next. Each well contains 200 .mu.l of the cZE
separation buffer. This process is repeated with samples from the
other fractions collected during CIEF.
[0179] Samples from CZE fractions are further resolved by CGE.
Fractions from CZE are separately concentrated by rotary
evaporation to a final liquid volume of about 5 .mu.l. The protein
sample is isolated from crystallized urea by refrigerated
(4.degree. C.) centrifugation. Ten microliters of SDS sample buffer
is added to each vial of protein concentrate. The SDS sample buffer
consists of 100 .mu.l of eCAP SDS sample buffer (Beckman Coulter,
Cat # 241525), 10 .mu.l of eCAP Orange G Reference Marker (Beckman
Coulter, Cat # 241524), and 90 .mu.l of anhydrous glycerol.
[0180] Each sample is run in cGE mode using a linear
poly(acrylamide)-coated fused silica capillary 60 cm long with a
100 .mu.m internal diameter. Commercially available eCAP SDS 14-200
Gel buffer (Beckman-Coulter Cat # 477416) is used for the
separation and included in both reservoirs. The separation is
conducted at 20.degree. C. and 500 V/cm for 50 min. Molecular
weight calibration is conducted in a separate run using eCAP MW
Standards (Beckman-Coulter Cat # 477418) as described by the
manufacturer. A 100 sec sample injection at 0.5 psi is used to load
each sample into the capillary. Resolved proteins are detected by
fluoroscein fluorescence with a 466 nm laser induced fluorescence
detector.
[0181] The foregoing process is repeated with multiple samples from
diseased and control subjects, as well as replicate runs with
samples from the same subjects. The results are then examined to
identify proteins whose relative abundance varies between diseased
and control subjects. Such proteins are potential markers for the
particular disease and/or a drug target or potential drug.
[0182] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all purposes
to the same extent as if each individual publication, patent or
patent application were specifically and individually indicated to
be so incorporated by reference.
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References