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SCIENCE WATCH |
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.
Correspondence to: Dr. Mark A. Knepper, National Institutes of Health, Bldg. 10, Room 6N260, 10 Center Dr. MSC 1603, Bethesda, MD 20892-1603. Phone: 301-496-3064; Fax: 301-402-1443; E-mail: knep{at}helix.nih.gov
Abstract
ABSTRACT. Proteomics can be defined as "the systematic analysis of proteins for their identity, quantity, and function." The central concept is "multiplexing," i.e., simultaneous analysis of all proteins in a defined protein population, rather than investigation of one protein at a time, as in traditional biochemistry. Two major approaches have been described: (1) mass spectrometry-based approaches and (2) protein micro-array approaches. The purpose of this Science Watch article is to describe the fundamental features of these two approaches and to speculate on how proteomics will be useful in nephrology and nephrology research in the coming years.
According to Peng and Gygi (1), proteomics can be defined as "the systematic analysis of proteins for their identity, quantity, and function." It differs from protein chemistry or biochemistry in its wide scope, investigating populations of proteins rather than one protein at a time. Proteomics is a rapidly growing field whose development has been a major spin-off of the human genome project and genome sequencing projects for other living species. It would be advantageous for renal physiologists and nephrologists to be familiar with the major methodologies that are under development because of the great potential that proteomics approaches have for the study of renal physiology and pathophysiology and because of the potential applications of proteomics in clinical medicine. The purpose of this Science Watch article is to summarize these technologies and illustrate how they will be useful in "discovery" research in the future. I do not attempt to comprehensively review the topic, but instead provide selected references that highlight critical technical developments.
Why Study Proteins?
Before discussing proteomics methodologies, it is informative to consider the rationale for studying proteins rather than nucleic acids for first-level investigation of regulatory and adaptive phenomena in biologic tissues and analyses of body fluids. First, one must recognize that proteins, rather than nucleic acids, mediate most of the physiologic functions within the cell. Hence, if one is interested in physiology or pathophysiology at a cell, tissue, or organ level it makes sense to study the prime mediators of function, the proteins. Second, analysis of body fluids such as urine (2–4) can only be accomplished by proteomics approaches because nucleic acids play no direct functional role in extracellular fluids. Third, one must recognize that proteins are regulated in a multiplicity of ways, many of which do not involve changes in mRNA levels. As summarized in Table 1, protein abundance can change as a result of altered transcription or mRNA stability, but also as a result of direct regulation of translation or regulation of protein half life. Additional regulatory mechanisms do not depend on changes in protein abundance but rather on modification of proteins by proteolytic processing, post-translational modifications, regulated trafficking, or protein-protein interaction. Proteomic methods are under development for the large-scale study of all of these modes of regulation, providing information extending far beyond transcriptional regulation. It is clear, therefore, that most forms of cellular regulation would not be detectable by cDNA or oligonucleotide arrays. Thus, the field of proteomics can be viewed as being complementary to the area of functional genomics.
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Applications of proteomics methodologies are also of growing importance in drug discovery tasks (9), largely because drugs may act by mechanisms manifested at the level of protein function rather than through changes in gene transcription. Furthermore, adverse effects of drugs in many cases results from direct interaction with proteins, without direct manifestations at a nucleic acid level.
To summarize, there are several reasons to focus on large-scale study of proteins (rather than nucleic acids) if the objective is to understand organ physiology or pathophysiology or to pursue drug discovery tasks. I turn now to a more formal consideration of proteomics methodologies.
Proteomics Methodologies: General Considerations
A practical approach to proteomics analysis involves simultaneous analysis of all members of a defined set of proteins. The common element of proteomics studies is "multiplexing," i.e., the simultaneous study of multiple proteins rather than one protein at a time, as in traditional biochemistry.
Initially, proteomics has been viewed as an element of the field of functional genomics. Because technologies for detecting and separating proteins are necessarily much different from methods applied to nucleic acid analysis, the two fields have evolved in somewhat different directions. In particular, because the physiochemical properties of proteins are so highly variable, the goal of looking at all proteins simultaneously is much more distant than analysis of all transcripts simultaneously. Consequently, most proteomics-based investigations focus on defined subpopulations of proteins. Therefore, the first step in a proteomics study is to define a protein population to be investigated. Such a population could be, for example, the set of all proteins found in urine or blood plasma, the set of proteins present in endosomes from collecting duct principal cells, the set of all proteins that form complexes with the proximal tubule Na-H exchanger NHE3, the set of all Na transporters expressed along the renal tubule, or the set of all renal cortical proteins detectable in two-dimensional gels by Coomassie staining. Obviously, the subpopulation of proteins to be investigated is a function of both the interests of the investigator and the limits of technology.
In general, proteomics can be divided into two broad areas based on the detection methods used: (1) approaches using mass spectrometry to detect and identify proteins and (2) approaches using arrays or ensembles of binding molecules to detect and identify proteins. The latter approach most commonly utilizes antibodies as the binding molecules. In the remainder of this Science Watch article, I will summarize the key features of these two methodologies.
Proteomics Met2hods Based on Mass Spectrometry
In general, proteomics methods based on mass spectrometry depend on a separations step followed by mass spectrometry to identify the proteins of interest. Currently, the most common separations step is two-dimensional electrophoresis.
Two-Dimensional Electrophoresis
Figure 1 shows an example of a silver-stained gel from two-dimensional electrophoresis of a kidney homogenate (10). In two-dimensional electrophoresis, the first-dimension separation is usually achieved by isoelectric focusing, which stratifies proteins on the basis of their isoelectric points. The second step is standard electrophoresis, which stratifies the proteins on the basis of molecular weight. After staining with Coomassie blue dye, proprietary fluorescent dyes, or other methods, individual spots of interest can be cut out of the gel and identified by mass spectrometry. Various schemes have been devised to identify spots of interest on the basis of differential expression or post-translational modifications, for example. These variations are discussed below.
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Because of these limitations of two-dimensional electrophoresis, there is presently strong movement in the direction of alternative separations techniques, such as liquid chromatography and capillary electrophoresis upstream from the mass spectrometer (1).
Figure 2 shows a simple flow chart for identification of proteins of interest on a two-dimensional electrophoresis gel. A critical intermediate step is digestion of the protein spot using a protease, almost always trypsin. Most proteins are too large to be unequivocally identified intact by mass spectrometry. Trypsin cleaves polypeptide chains at K-X or R-X sites, where K is lysine, R is arginine, and X is any amino acid other than proline. Trypsin digestion breaks a given protein into a unique series of peptide fragments. These fragments are identified by mass spectrometry.
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Peptide Mass Fingerprinting
Peptide mass fingerprinting identifies a protein by measuring the molecular masses of all major trypsin products and matching these molecular masses with databases of theoretical sizes of trypsin fragments from known protein sequences (Figure 3). Because trypsin cleavage sites are predictable (cleavage after lysines and arginines), a known protein sequence can be readily converted to a unique set of peptide masses by computer analysis. Peptide mass fingerprinting is usually accomplished using MALDI-TOF technology.
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In most tandem mass spectrometers, the method for creating gas phase ions at the input to the mass spectrometer is electrospray ionization (13). With this technique, an acidic, aqueous solution that contains the peptides is sprayed through a small diameter needle. A high positive voltage is applied. Because the peptides have been acidified, they are positively charged and the voltage propels them toward the mass spectrometer mass analyzer.
Applications of Mass Spectrometry-Based Proteomics
Mass spectrometry-based proteomics approaches can be used in a conceptually simple way to survey a given tissue or organelle to identify its proteome, i.e., generate a list of expressed proteins. However, the most promising applications in future years will be for large-scale identification of regulatory processes manifested at a protein level. Table 1 summarizes major modes of regulation involving proteins.
Abundance Profiling.
Great interest currently exists in detection of changes in protein abundance in a given tissue in response to a given physiologic or pathophysiologic perturbation. Conceptually, the simplest way to do this would be to isolate protein extracts from the two samples and compare silver- or Coomassie-stained two-dimensional gels to identify proteins whose abundance has changed. An early success of this type of approach utilized comparative analysis of two-dimensional gels to identify proteins whose expression is increased or decreased in cycloporine A nephrotoxicity (16). Such comparisons are often difficult because of gel-to-gel variability, requiring complex algorithms to manipulate gel images so that they are in precise alignment. One solution to this problem is to derivatize the experimental and control samples with different labels so that the samples can be mixed and analyzed, allowing direct relative quantification in the same gel. One such approach, difference gel electrophoresis (DIGE) (17), is illustrated in Figure 6. Here, two protein samples (a control and an experimental sample) are derivatized using reagents that attach different fluorescence dyes, Cy3 and Cy5, to lysines in the protein molecules. The derivatizing reagents are matched so that they do not cause differential changes in isoelectric point or molecular weight. This allows the samples to be mixed and run on the same two-dimensional electrophoresis gel. The finished gel can be imaged at the wavelengths appropriate for the two dyes and analyzed by computer to detect protein spots whose abundances have been altered by the experimental manipulation. These differentially expressed proteins can be excised from the gel, trypsinized, and identified by mass spectrometry. In principle, the DIGE technique could also be used to investigate post-translational modifications when these modifications lead to detectable changes in protein isoelectric point and molecular mass.
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Post-Translational Modifications.
In addition to studies of protein abundance, mass spectrometry can be used for large-scale investigations of protein modifications. Of considerable interest is large-scale detection of post-translational modifications, including phosphorylation, acylation, ubiquitination, glycosylation, etc. The greatest progress has been made in development of techniques to investigate targets for protein phosphorylation. The most fundamental approach is to label proteins from experimental and control samples with 32P and then to compare autoradiograms of two-dimension gels to identify proteins spots in response to the experimental manipulation. This approach has been successful but can misidentify phosphorylated proteins if they overlie more abundant related proteins. Therefore, success with this method requires confirmation of the phosphorylation by tandem mass spectrometry or other approaches. An alternative approach is to carry out an initial step that enriches the phosphorylated proteins in the sample. This can be done, for example, by immunoprecipitation with phosphorylation-specific antibodies before mass-spectrometric identification. Although this approach works well for identification of proteins phosphorylated on tyrosines, it has not been successful for isolation of proteins phosphorylated on serines and threonines. Consequently, alternative techniques have been developed for affinity isolation of phosphorylated proteins by covalent attachment of affinity tags at sites of phosphorylation (18,19).
Protein-Protein Interactions.
Cell function is dependent on a myriad of protein-protein interactions. One approach to the identification of protein-protein interactions that has been widely employed is the yeast two-hybrid method (20). With this technique, two yeast strains containing two different cDNA libraries are mated. One has its open reading frames fused with the DNA-binding domain of GAL4, and the other has its open reading frames fused with the activation domain of GAL4. A GAL4-dependent reporter gene is expressed only when the DNA-binding and activation domains of GAL4 are brought together by binding of the proteins to which they are fused. The DNA inserts from the interacting plasmids are sequenced to identify the interacting proteins. This technique was employed, for example, to identify Nedd4 as a binding partner for the aldosterone-regulated epithelial sodium channel ENaC (21). Nedd4 is believed to decrease the protein half-life of ENaC in the kidney by ubiquitinating one or more ENaC subunits, a process that has been demonstrated to be downregulated by the aldosterone-activated kinase sgk (22,23). Recently, the yeast two-hybrid method has been employed in a large-scale effort to comprehensively map pair-wise protein interactions in yeast (24,25). This has yielded thousands of putative interactions, opening the way for large-scale systems modeling to address the physiologic significance of these interactions in the context of the whole organism.
One limitation of the yeast two-hybrid approach is that it only identifies binary interactions. Many or most protein complexes consist of more than two proteins; therefore, alternative approaches have been under development to study multiprotein complexes. One such approach is to affinity purify multiprotein complexes and identify the members of the complex by mass spectrometry. For example, this can be done by immunoprecipitation or by affinity isolating proteins-of-interest after expressing them as fusion proteins that include an affinity tag such as glutathione S-transferase (GST), which allows the fusion proteins and their binding partners to be fished out of the protein mixture using glutathione bound to a solid substrate as agarose beads. Proteins present in affinity-isolated complexes can then be identified by mass spectrometry. Mass spectrometry-based identification of protein-protein interactions have begun to be carried out at large scale in yeast, identifying thousands of potential protein-protein interactions (26,27). One drawback of this approach is that protein overexpression in cells may alter binding patterns relative to those present at the native level of expression.
Proteomics Methods Based on Ensembles or Arrays of Binding Proteins
A widely heralded new approach to proteomic analysis is the use of ensembles or arrays of binding proteins (or other macromolecules) to identify individual proteins in complex protein mixtures (28). Antibodies represent the most common class of binding protein chosen for such approaches and will be emphasized here. Ensembles of antibodies can be chosen to identify known members of certain populations of proteins in a "targeted proteomics" approach. Examples include antibodies to proteins involved in cancer-related signaling (29), antibodies to proteins involved in cell cycle regulation and apoptosis (30), and antibodies to sodium transport proteins expressed along the renal tubule (31,32). Alternatively, large numbers of affinity-matched antibodies can be obtained from recombinant sources such as phage display libraries. Such antibody ensembles can provide broad coverage of epitopes found in large populations of proteins, allowing individual proteins to be identified by matching patterns of epitope recognition. I will describe these two approaches in turn.
Targeted Proteomics Approaches
Over the years, physiologic and biochemical investigations have identified the major proteins involved in physiologic processes, such as glucose metabolism, cell cycle regulation, growth factor receptor signaling pathways, and salt and water transport along the nephron. Cloning of cDNAs for these proteins has made it possible to produce high-quality polyclonal or monoclonal antibodies to them, using synthetic peptides as immunogens, obviating the need for protein purification. Facile production of antibodies has allowed investigators to accrue comprehensive sets, or "ensembles," of antibodies against proteins relevant to a given physiologic process. These ensembles of antibodies allow integrated investigation of entire pathways through simultaneous assessment of the regulatory state of all members of the pathway. An example of this approach was the use of an ensemble of antibodies to all of the major Na transporters expressed along the nephron to screen renal homogenates from rats undergoing mineralocorticoid escape to determine which transporters are downregulated to account for the associated increase in Na excretion (33). The technical approaches used to detect changes in abundance of proteins have included multiplexed immunoblots and antibody micro-arrays. I discuss these two approaches in the next two subsections.
Multiplexed Immunoblots.
If the number of proteins in a targeted protein population is relatively low (<20), it is advantageous to assess abundance changes through quantitative or semiquantitative immunoblotting (31). Semiquantitative immunoblotting (with proper loading controls) allows precise simultaneous comparison of multiple samples, whereas antibody micro-arrays generally permit only binary comparisons. We have employed this approach to carry out a systemic identification of renal tubule transporters that are targets for adaptive regulation by hormonal and non-hormonal regulatory factors involved in the control of BP and extracellular fluid volume (34,37). An example of the approach applied to detection of transporter targets for regulation by aldosterone is shown in Figure 7 (35,38). Additional studies using the same ensemble of antibodies have identified Na transporters dysregulated in animal models of various disorders of NaCl and water balance, including chronic renal failure (39), ischemia-induced acute renal failure (40), cirrhosis (41), lithium-induced nephrogenic diabetes insipidus (42), syndrome of inappropriate antidiuretic hormone secretion (43), primary aldosteronism (33), and vitamin D–induced hypercalcemia (44).
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Antibody Micro-Arrays.
Targeted analysis of protein expression can also be carried out using antibody micro-arrays consisting of large numbers of antibodies deposited as discrete spots on a solid substrate (28). An approach to identification of differentially expressed proteins in two complex mixtures of proteins using the antibody micro-array concept was reported by Haab et al. (47). In this method, described in Figure 8, lysine moieties in two protein samples were derivatized with two different fluorescence dyes, Cy3 and Cy5, using N-hydroxysuccinimide-ester–activated dyes. The samples were mixed and exposed to a micro-array containing 115 antibodies spotted onto poly-L-lysine–coated glass slides with a robotic micro-arrayer. The fluorescence signal associated with the bound proteins was quantified using a standard micro-array reader, allowing signal normalization and calculation of relative protein abundances for the two samples for each of the proteins on the array.
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In principle, antibody array experiments can be carried out like cDNA array and oligonucleotide array experiments, revealing broad patterns in protein expression in mammalian tissues. However, the goal of developing very large antibody chips with comprehensive coverage of mammalian proteomes will be much more difficult to achieve than was production of chips for mRNA expression studies. This circumstance is in part owing to the fact that proteomes are much larger than transcriptosomes from the same tissue, owing to the variety of protein modifications that occur. Thus, more array features are needed than with DNA arrays. The major barrier, however, is the technical approach to antibody production. The conventional means of antibody production, through immunization of animals, is expensive and time consuming. The antibodies produced are highly variable in affinity and selectively, making it difficult to choose a single condition for antibody micro-array incubations that will optimize antibody-binding reactions for all the antibodies spotted on an array. A solution to this problem will be seen in the next few years, however, as methods for production and screening of recombinant antibodies are perfected.
Recombinant Antibodies.
Methods have been described for large-scale production of recombinant antibodies and antibody fragments in plants ("plantibodies") (48,49) and in bacteriophage (using "phage-display" techniques) (50,51).
Phage-display antibody libraries are produced though random recombination of IgG heavy and light chain variable region genes, an approach that allows production of antibodies recognizing millions of epitopes. Individual bacteriophage clones can be selected to obtain recombinant antibodies that crossreact with mammalian tissue proteins. Antibody clones with uniform affinities for their target epitopes can be selected for spotting onto arrays, allowing easier optimization of antigen binding throughout the array. An apparent drawback is the need to prepare large numbers of purified proteins or peptides to screen bacteriophage clones to identify recombinant antibodies that recognize specific proteins.
In practice, the need for identification of the target proteins for each recombinant antibody may be bypassed for some types of applications, such as drug discovery and disease screening. For such arrays, selection of antibodies may be based on recognition of unknown proteins present in complex protein mixtures from a given tissue, rather then on recognition of specific proteins. Such arrays will therefore be most useful for identification of patterns of response that may correlate with specific pathologic processes or drug responses, rather than as a means of identifying specific responding proteins. Computer analysis of patterns of response on antibody micro-arrays built from recombinant antibody sets could therefore provide an empirical basis for drug discovery, diagnosis of disease, and monitoring of therapy. Commercial development of methods for creating ensembles of recombinant antibodies is underway at a number of sites throughout the world, making it likely that practical large-scale antibody chips will be available for routine application within the next few years.
Conclusion
Proteomics methodologies, both mass spectrometry-based approaches and protein-chip approaches, hold great promise in basic renal research and in clinical nephrology. Over the past ten to fifteen years in the basic research arena, research has focused on identification of individual genes and proteins and characterization of protein function at a molecular level. It is clear, however, that complex cellular function is dependent on networks of proteins that interact and are co-regulated. Identification of these networks will depend on development and application of high-throughput protein analysis such as described in this paper. In the area of clinical nephrology, there is great reason for optimism about the applicability of proteomics methodologies in disease diagnosis and therapeutic monitoring. One important application will be to screen urine samples for early detection of genetic or acquired renal diseases. Recent studies have demonstrated that the urinary proteome contains a much greater variety of proteins than previously recognized (2–4,52,53), and it is likely that analysis of changes in the relative abundances of urinary proteins will be informative. With regard to clinical proteomics in tissues, the greatest efforts are currently focused on characterization and staging of various neoplasms (54), including renal cell carcinomas (10). Application to renal biopsy material may be on the horizon, as well, if limitations of sensitivity and tissue heterogeneity can be faced.
Acknowledgments
This work is funded by the Intramural Budget of the National Heart, Lung, and Blood Institute (National Institutes of Health, project no. Z01-HL-01282-KE to M. A. Knepper).
Footnotes
Based on a lecture given at a Workshop on Genomics at the Annual Meeting of the High Blood Pressure Research Council of the American Heart Association, September 22, 2001.
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