THEME
Cutting-Edge Technology
II. Proteomics: core technologies
and applications in physiology
Frank A.
Witzmann and
Junyu
Li
Departments of Cellular and Integrative Physiology and Biochemistry
and Molecular Biology, Indiana University School of Medicine,
Indianapolis, Indiana 46202
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ABSTRACT |
Technologies for proteomics, e.g., studies
examining the protein complement of the genome, have been in
development for over 20 years. More recently, proteomics has become
formalized by combining techniques for large-scale protein separation
with very precise, high-fidelity approaches that analyze, identify, and
characterize the separated proteins. These methods bring to reality the
powerful scope of proteomics, enabling researchers to investigate
cellular function at the protein level and thus representing one of
proteomics' most fitting applications. In this review, we take a brief
and concise look at some of the current, physiologically relevant technologies that comprise proteomics and report specific applications in which proteomics has provided valuable biological insight.
protein analysis; two-dimensional electrophoresis; mass
spectrometry; isotope-coded affinity tags
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INTRODUCTION |
THE BROAD RANGE OF MOLECULAR
mechanisms that governs cellular function is largely administered via
the structure and function of genetically encoded products, the
proteins. Collectively, these gene products represent the proteome, and
their analysis has come to be known as proteomics. The actual number of
functionally unique protein types in the human proteome variably
expressed across assorted human cell types from the >30,000 available
genes is estimated to be 100,000. With multiply-modified forms of each, that number could approach a million. This diversity is the result of
widespread posttranscriptional processing of mRNA and co- and posttranslational processes. Both of these lead to a fair degree of
discordance between the open reading frames predicting protein structure and the actual functional product. Consequently, a full understanding of function, disease processes, and clinical intervention necessitates expression analysis at the protein level. Additionally, the range of fully functional protein abundance in a cell may reach
nine orders of magnitude. Proteomics thus presents investigators with a
daunting technological task, both in terms of protein identification and quantification. Although originally designated as a global approach
to identify the entire proteome (34, 36), using
two-dimensional (2D) electrophoretic (2DE), mass spectrometric, and
bioinformatic techniques, proteomics has become a diverse science that
includes nearly all manner of separation, affinity purification, and
protein chemistry components.
Considerable effort has been and continues to be placed on removing the
technical barriers that impede proteomic efforts. We now understand
both the strengths and limitations of the "first generation"
proteomics approaches capable of generating significant biological
insight, yet generally providing narrow data (protein presence/absence,
protein identification) for high and moderately abundant proteins
(6). This realization has led to expanded development and
implementation of chromatographic separation techniques, improved mass
spectrometry (MS), automation via robotics, and growth of
multidimensional biomolecular datasets (e.g., posttranslational modifications, subcellular localization, protein interactions, protein
abundance, and protein function). Further technological developments
will continue to drive forward the next generation of proteomic
techniques and approaches. These developments have widened the scope of
proteomics and have fueled the explosion of interest in this field. Two
full issues of Trends in Biotechnology have addressed these
developments in detail (4, 37), and readers are encouraged
to consult them. This themes article highlights some of the
technologies central to contemporary proteome analysis and provides
examples of how these have been applied to physiological questions.
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DIFFERENTIAL EXPRESSION PROTEOMICS |
2DE and MS.
As mentioned above, proteomics originated as a direct result of
technical developments in 2DE protein separation and MS instrumentation and the explosion of genome sequence information that generated protein
sequence databases. Often referred to as "peptide mass fingerprinting," this first-generation, or "blue-collar,"
proteomics approach is still the most commonly used proteomics strategy
and the most practical and economical for academic laboratories.
In 2DE, proteins are subjected to orthogonal separation methods; the
first based on protein charge via isoelectric focusing (IEF) and then
by mass in sodium dodecyl sulfate PAGE. The relatively recent
development of immobilized pH-gradient gel (IPG) strips to improve
first-dimension IEF separations shows promise, although gel-based IEF
remains a useful tool for the patient and resourceful. The final
product of 2DE separation is essentially an in-gel array of proteins,
each assuming a coordinate position corresponding to the unique
combination of isoelectric point (pI) and mass. Resulting 2D protein
patterns are visualized by a number of methods: visible and/or
fluorescent dyes, silver stains, or autoradiography. Typically, scanned
gel images are analyzed by any of a number of ever-improving 2D gel
analysis software packages. It is here that both the strengths and
weakness of this approach become evident. Protein abundance comparisons
(e.g., differential expression) are easily made, because differences in
protein spot density are readily detectable and can be quantified
robustly and compared statistically. However, unless one conducts
highly parallel 2DE runs, gel-to-gel variation becomes problematic and
image analysis an exercise in frustration.
Despite the insightful design and implementation of parallel 2DE nearly
24 years ago (2, 3) and numerous examples of its utility
in differential protein expression analyses across a large number of
samples, surprisingly, this approach has not been used widely. Unlike
trends in 2D gel analysis software that enable the concurrent analysis
of hundreds of gel patterns per experiment, electrophoretic equipment
manufacturers have lagged behind. Although efforts have been made to
address the technical necessity of highly parallel 2DE by scaling the
process up to 12 gels/run maximum (e.g., Bio-Rad, Amersham Biosciences,
etc.), contemporary 2DE instrumentation still falls short of the scale necessary (>20-24 2D gels/run). Figure
1 illustrates this point by presenting a
montage of multiple patterns from a single 2DE experiment. Here, 36 individual 2D gels were run (20/run) analyzing 36 individual wells from
six 6-well culture plates on which human keratinocytes were cultured.
Parallel analysis of this type makes differential expression analysis
robust and simplifies candidate protein selection.

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Fig. 1.
Example of gel-to-gel consistency across 36 individual
gel patterns representing 36 individual human keratinocyte samples
incubated in six 6-well plates and solubilized on the plates (medium
removed). Samples were separated by 2-dimensional (2D) electrophoresis
(2DE), 20 gels/run in the authors' laboratory; thus results from 2 separate runs are shown. The first (top left) pattern is the
reference pattern in PDQuest analysis, the next 12 patterns are
controls, and the remaining 24 are jet fuel exposed. Highly parallel
2DE is essential for inclusion in successful differential expression
proteomics studies.
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A recent development that addresses gel-to-gel variability using 2DE
incorporates sensitive fluorescent protein staining and can be achieved
without extensive parallel 2DE instrumentation is 2D differential gel
electrophoresis (DIGE) (32). By using replicate gels of
pooled samples separated on single 2D gels from multiple individual
animals (control vs. treated in each gel) (31), this
approach has been used and validated to determine quantitative protein
differences in acetaminophen toxicity. In this clever approach, control
and treated samples are labeled with Cy3 or Cy5 dyes and mixed before
application onto the same 2D gel. As a result, the same form of a given
protein from each sample will migrate to the same position on the 2D
gel. The relative abundance of each protein in each sample is then
obtained by scanning the gel using excitation and emission wavelengths
unique to each Cy dye.
After 2DE separation and image analysis, any or all of the proteins in
the gel pattern can be selected for identification. In large, 2D
gel-based projects, the ultimate goal is to identify all resolved
proteins. In less ambitious studies, only those proteins differentially
expressed are of interest. Regardless of project scope, the general
approach to protein identification is to cut the protein spot from the
gel and digest it with a proteolytic enzyme such as trypsin. The
resulting digest mixture is then analyzed by matrix-assisted laser
desorption ionization (MALDI) time of flight MS (26). The
measured and optimized monisotopic mass data are then compared with
theoretically derived peptide mass databases, generated by applying
specific enzymatic cleavage rules to predicted/known protein sequences.
Whereas MALDI-based peptide mass fingerprinting enables
high-throughput, accurate, and sensitive mass detection and may result
in a large percentage of convincing protein identifications, many are
ambiguous and require confirmation. Frequently, some digested proteins
remain completely unidentified, despite yielding measurable peptides.
For unambiguous identification of 2D separated proteins, "peptide
sequence tag" data derived by MS/MS (27) (with either MALDI or electrospray ionization ion sources) can be compared with
expressed sequence tag databases, ever-expanding sources of
genomic/proteomic information representing a number of organisms.
Because the dynamic range of protein expression in most whole cell or
tissue lysates is huge and only the most abundant proteins from 2D gels
can be analyzed (despite excellent mass spectrometer sensitivity), far
too many proteins are overlooked. Furthermore, hydrophobic proteins and
those with very alkaline pI are poorly resolved on conventional 2D
gels. Even in rarely employed very large format gels, no more than
10,000 proteins have been analyzed on a single gel. To overcome the
problems of sensitivity and scope, tissue/cell fractionation methods
are used to enrich the sample gels with organellar proteins, and as a
result, several organellar proteomes are being characterized (9,
15, 19, 30).
Narrow-range IPG strips in 7-, 11-, 17-, or 24-cm lengths can bracket
the pH range of first-dimension separations extending to fairly
alkaline pH. Using these strips represents another significant step in
overcoming the limits of 2DE by significantly expanding the resolving
power of otherwise broad-range IEF. Separations achieved in narrow pI
windows greatly increase resolution and provide access to proteins that
are either undetectable or comigrate when separated using a broad-range
pH gradient. This approach thus becomes integral to any attempt to
analyze thousands of sample proteins, particularly for those
low-abundance proteins that would otherwise remain undetected. In this
regard, "virtual" gel patterns, such as those described by Cordwell
et al. (5), significantly increase the number of proteins
resolved (Fig. 2) and represent an
important technical development. Finally, significant improvements in
protein-detection sensitivity have been achieved by incorporating fluorescent and MS-compatible silver stains (22, 24, 29). In combination, these technical advances continue to make 2DE an
important component of the proteomics arsenal.

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Fig. 2.
Schematic view of subproteome approach. Cells and tissues are
initially prefractionated into cellular compartments or relative
protein solubilities. The samples are screened using wide-range 2D gels
to determine sample complexity and then, if necessary, passed through
higher resolution 2D to map low-abundance proteins, efficiently
separate overlapping proteins, and to map proteins to their cellular
location. Modified from Ref. 5.
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For example, 2DE, fluorescent staining (2D-DIGE), image analysis, and
peptide mass fingerprinting were combined recently to analyze the
cardiac mitochondrial proteome in murine creatine kinase (CK)
double-knockouts (KO) (20). Proteomic analysis
demonstrated that despite the absence of all isoforms of CK in the KO
mice, the cardiac mitochondrial proteome was identical to wild types, and, more importantly, its consistency mirrored a lack of altered cardiac function. Similarly, this approach was used recently to study
human bronchial biopsy samples. Proteins from these samples, which
correlated to the transformation of normal fibroblasts to myofibroblasts, were quantified and identified during the remodeling processes observed in asthma (35). Large-scale
protein database development for toxicologic/pharmacological
applications is an ongoing enterprise in many industrial laboratories.
This is typified by Fountoulakis' 2D gel-based mouse liver protein
database (8) that lists hundreds of identified proteins
screened against acetaminophen and cites numerous other databases
designed for general toxicologic screening applications.
Isotope-coded affinity tags.
An emerging approach that directly addresses the dynamic range and
solubility limitations of 2DE combines the separating power of liquid
chromatography (LC) with the highly accurate and sensitive mass
detection of tandem MS (13). Isotope-coded affinity tags (ICATs) are reagents containing a cysteine-reactive group, a linker with either eight hydrogens (light) or deuteriums (heavy), and a biotin
(affinity) moiety. As shown in Fig. 3, by
reacting each with light or heavy ICAT, relative protein abundance
comparisons between two different cell states can be made. The proteins
from each sample are combined and proteolytically digested, tagged peptides are collected by affinity chromatography, peptides are analyzed via LC-MS for relative quantitation of the isotopes on identical peptides, and finally, peptides are analyzed by
LC-MS/MS for protein identification. Despite limitations of its
own, ICAT technology is being improved (28) and has
already proven its utility in functional studies. For example,
characterization of analytically troublesome lipid-raft proteins has
been simplified (33), the proteomic components of a
complex cellular metabolic pathway have been studied in the context of
their functional genomic elements (17), and proteins of
subcellular microsomes have been identified and quantified in
differentiating human myeloid leukemia (HL-60) cells (14)
using the ICAT approach. The latter investigation included a common
addition to contemporary proteomic approaches, e.g., multidimensional
chromatographic separation of complex peptide mixtures. In this case,
sample complexity was reduced by subjecting isotopically labeled
proteolytic peptide mixtures to cation-exchange chromatography,
avidin-affinity chromatography and reversed-phase HPLC before automated
mass spectrometric characterization.

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Fig. 3.
Schematic representation of the isotope-coded affinity tag method.
The cysteine side chains in the complex mixtures of proteins from 2 different cell states are reduced and alkylated using the day
0 (d0)-labeled tag for the proteins in 1 cell state and the
day 8 (d8) form of the tag for the proteins in the second
cell state. The 2 mixtures are then combined and subjected to a
proteolytic digestion. The resultant complex mixture of proteolytic
peptides is purified with an avidin column to pull out only the subset
of labeled peptides via the affinity tag. Quantitation of differential
expression is based on the relative abundance of the isotopes in the
mass spectrometry spectrum. Modified from Mosely MA,
Trends Biochem Sci, Suppl 19, S11, 2001.
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Because an estimated 20% of the human proteome includes proteins
lacking at least one cysteine residue, alternatives to cysteine-based labeling that can tag every protein are being developed. For instance, a clever variation of the ICAT approach has been developed to identify
and quantitate the extent of protein phosphorylation using a
phosphoprotein isotope-coded affinity tag (PhIAT) (12). The PhIAT methodology is similar to the ICAT approach in that it
enables proteome-wide purification and quantitation of peptides containing specific types of residues; in this case phosphopeptides. Its potential in the characterization of cellular signaling is promising.
To both simplify complex peptide mixtures mentioned above and to target
specific subgroups of related proteins and selectively identify them, a
strategy using "signature peptides" (10, 18) isolated
by an array of elaborate yet automated affinity and chromatography separations is being developed to study global phospho- and
glycoprotein expression (11, 25). These multidimensional
separation approaches represent an effective trend in proteomics
(independent of ICATs), and their implementation should prove
particularly useful in physiologically directed differential expression
and qualitative proteomics studies.
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FUNCTIONAL AND STRUCTURAL PROTEOMICS |
Many protein-mediated cellular functions are managed and regulated
by mechanisms that do not involve quantitative changes in expression.
Instead, they are the consequences of qualitative modification of
existing proteins, chemical additions such as phosphorylation,
glycosylation, and lipidation, or modifications such as oxidation and
deamidation. Second, proteins that mediate most cellular processes
function as constituents of macromolecular complexes, not as individual
entities acting independently. The proteomic approaches described thus
far are rather reductionist. Clearly, these are useful ways in which to
study the proteome; however, to effectively study function,
investigators also must focus on protein-protein interactions and the
characterization of multimeric protein complexes. In this respect, two
areas in which proteomics is playing a significant role in our
understanding of cellular function are the characterization of
posttranslational modifications (functional proteomics) and protein
interactions (structural proteomics).
The phosphoproteome.
Given the importance of protein phosphorylation in the regulatory
activities of cellular function and the amplification of signaling
cascades that distinguish these activities from others, it is not
surprising that phosphorylation is the most common covalent protein
modification in mammalian cells. Indeed, the huge number of protein
kinases and phosphatases encoded by the genome underscore their
significance. Global analysis of the phosphoproteome has thus evolved
into an integral facet of physiology. Historically, phosphoproteins
were studied on Western blots using antiphosphoserine or
antiphosphothreonine antibodies. This approach is still adequate qualitatively but not quantitatively, because it suffers from the same
general limitations of 2DE mentioned earlier. In-gel digestion and
phosphopeptide analysis are deemed feasible but impractical
(21). As alternatives, recent analytical approaches to the
phosphoproteome incorporate either phosphopeptide enrichment using
metal affinity columns, phosphatase treatment before MS/MS, or the use
of protein chips (39). These approaches are necessitated by the low stoichiometry of protein phosphorylation, the fact that
phosphopeptides are generally detected with low efficiency or not at
all by MS. Also, the hydrophilic phosphopeptides may be eluted and
therefore lost in the void volume during reversed-phase peptide cleanup
for MALDI.
New methods are in use that combine chemical modification and affinity
purification for the characterization of serine and threonine
phosphopeptides (1, 23). These methods are generally based
on the chemical replacement of the phosphate moieties by affinity tags
(biotinylation) followed by trypsin digestion. The biotinylated
peptides are then enriched by affinity-isolation, analyzed by LC-MS/MS,
and the phosphorylated residues are identified by automated database
searching. This approach has widespread potential utility for defining
signaling pathways and control mechanisms that involve phosphorylation
or dephosphorylation of serine/threonine residues.
In a related development, Snyder and his colleagues
(40) have engineered a novel approach for high throughput
screening of protein kinase (PK) activities by overproducing all the
yeast PKs as glutathione S-transferase fusions and
covalently affixing them to a chip surface in microarray format.
With the use of [33P]ATP, it was discovered that
particular proteins are preferred substrates for particular PKs and
that many PKs prefer particular substrates. This approach has enormous
potential application in the study of mammalian and human PK systems.
Protein-protein interactions.
As Eisenberg et al. (7) has so aptly proposed, "a
protein is defined as an element in the network of its interactions," and, as such, each protein in living cells functions as part of an
extended web of interacting molecules. In this regard, a more holistic
(as opposed to global) analysis of the proteome incorporates ingenious
approaches that involve 1) affinity purification of protein
complexes, the electrophoretic separation of the components, their
tryptic digestion, and the identification of each element (16) or 2) centrifugation purification of cell
components, tryptic digestion of the protein constituents followed by
multidimensional liquid chromatography and tandem mass spectrometric
identification (38).
As an example of the first approach, Blackstock's group
(16) isolated the mouse brain
N-methyl-D-aspartate (NMDA) receptor multiprotein complex (NRC) and, by analyzing its components, provided information that strongly suggests that subsets of neurotransmitter receptors, cell-adhesion proteins, adapters, second messengers, and
cytoskeletal proteins are all organized together into a physical unit
comprising the signaling pathway. Furthermore, several novel features
of the NRC observed in this study provide valuable insight into the
physiological context of NMDA receptor-dependent synaptic plasticity.
In the second approach, over 100 proteins can be analyzed per run via
direct analysis of large protein complexes. Applied to the eukaryotic
ribosomal proteome, its constituent complex of ~80 unique proteins is
rapidly and sensitively characterized, and unique features are
identified. This process demonstrates considerable potential in
characterizing, as well as detecting alterations in, other functionally
relevant protein complexes in a variety of cell systems.
In summary, the various cellular proteomes are dynamic, and
fluctuations in their characteristic expression are central to their
role in physiological regulation, disease and injury, and their
response to chemical intervention. Without a doubt, it is therefore essential that we conduct both broad and directed analyses of
the proteome's individual protein components to understand the
molecular underpinnings of physiological function. We must work to make
certain the technologies supporting such analyses continue to improve,
in turn, to ensure that the boundaries to our understanding disappear
as a result. Despite the limitations of current proteomics technology,
there exist a number of approaches from which to choose, specific for
each application. Whether one is interested in the differential
expression of a protein or group of proteins that underlie functional
alterations, posttranslational modification of resident proteins, or
the complex constituency and function of huge multiprotein complexes,
the tools are available, and they are improving.
This review has presented a limited sample of the many proteomic
approaches and technologies relevant to the physiologist. A cursory
look at the published literature by the reader will quickly demonstrate
the utility of this approach in life science and the breadth in which
its analytical power has been and will continue to be applied.
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FOOTNOTES |
Address for reprint requests and other correspondence: F. A. Witzmann, Depts. of Cellular and Integrative Physiology and
Biochemistry and Molecular Biology, Indiana Univ. School of Medicine,
635 Barnhill Dr., MS405, Indianapolis, IN 46202 (E-mail:
fwitzman{at}iupui.edu).
First published January 9, 2002;10.1152/ajpgi.00510.2001
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