For most of the twentieth century, the study of proteins was restricted primarily to the analysis of individual proteins. For example, one would study an enzyme by determining its enzymatic activity (its substrates, products, rate of reaction, requirement for cofactors, pH, etc.), its structure, and its mechanism of action. In some cases, the relationships between a few enzymes that participate in a metabolic pathway might also be studied. On a broader scale, the localization and activity of an enzyme would be examined in the context of a cell or tissue. The effects of mutations, dis eases, or drugs on the expression and activity of the enzyme might also be the subject of investigation. This multipronged approach provided deep insight into the function and mechanisms of action of individual proteins or relatively small numbers of interacting proteins. However, such a one-by one approach to studying proteins does not readily provide a global picture of what is happening in the proteome of a cell, tissue, or entire organism.
Proteomics Is the Study of All or a Large Subset of Proteins in a Biological System
The advent of genomics (sequencing of genomic DNA and its associated technologies, such as simultaneous analysis of the levels of all mRNAs in cells and tissues) clearly showed that a global, or systems, approach to biology could provide unique and highly valuable insights. Many scientists recognized that a global analysis of the proteins in biological systems had the potential for equally valuable contributions to our understanding. Thus a new field was born—proteomics. Proteomics is the systematic study of the amounts, modifications, interactions, localization, and functions of all or subsets of proteins at the whole-organism, tissue, cellular, and subcellular levels.
A number of broad questions are addressed in proteomic studies:
● In a given sample (whole organism, tissue, cell, subcellular compartment), what fraction of the whole proteome is ex pressed (i.e., which proteins are present)?
● Of those proteins present in the sample, what are their relative abundances?
● What are the relative amounts of the different splice forms and chemically modified forms (e.g., phosphorylated, methylated, fatty acylated) of the proteins?
● Which proteins are present in large multiprotein complex es, and which proteins are in each complex? What are the functions of these complexes, and how do they interact?
● When the state (e.g., growth rate, stage of cell cycle, differ entiation, stress level) of a cell changes, do the proteins in the cell, or those secreted from the cell, change in a characteristic (fingerprint-like) pattern? Which proteins change, and how (relative amounts, modifications, splice forms, etc.)? [An swering these questions requires a form of protein expres sion profiling that complements the transcriptional (mRNA) profiling discussed in Chapter 9.]
● Can such fingerprint-like changes be used for diagnostic purposes? For example, do certain cancers or heart disease cause characteristic changes in blood proteins? Can the proteomic fingerprint help determine if a given cancer is resistant or sensitive to a particular chemotherapeutic drug? [Proteomic fingerprints can also be the starting point for studies of the mechanisms underlying the change of state. Proteins (and other biomolecules) that show changes that are diagnostic of a particular state are called biomarkers.]
● Can changes in the proteome help define targets for drugs or suggest mechanisms by which a drug might induce toxic side effects? (If so, it might be possible to engineer modified versions of the drug with fewer side effects.)
These are just a few of the questions that can be ad dressed using proteomics. The methods used to answer these questions are as diverse as the questions themselves, and their numbers are growing rapidly.
Advanced Techniques in Mass Spectrometry Are Critical to Proteomic Analysis
Advances in proteomics technologies (e.g., mass spectrometry) profoundly affect the types of questions that can be practically studied. For many years, two-dimensional gel electrophoresis allowed researchers to separate, display, and characterize complex mixtures of proteins. The spots on a two-dimensional electrophoresis gel could be excised, the protein fragmented by proteolysis (e.g., by tryp sin digestion), and the fragments identified by MS. An alter native to this two-dimensional gel electrophoresis method is high-throughput LC-MS/MS. Figure 1 outlines the general LC-MS/MS approach, in which a complex mixture of proteins is digested with a protease; the myriad resulting peptides are fractionated by LC into multiple, less com plex fractions, which are slowly but continuously injected by electrospray ionization into a tandem mass spectrometer. The fractions are then sequentially subjected to multiple cycles of MS/MS until the sequences of many of the pep tides have been determined and used to identify the proteins in the original biological sample. Detection of a substantial fraction of the proteins in whole cells or tissues currently requires samples containing more than 50 μg of protein, an amount equivalent to the protein content of some 70,000 200,000 mammalian cells. Efforts are under way to increase the sensitivity of the method so that eventually one might be able to analyze the proteome of an individual cell.
Fig1. LC-MS/MS is used to identify the proteins in a complex biological sample. A complex mixture of proteins in a biological sample (e.g., an isolated preparation of Golgi organelles) is digested with a protease. The mixture of resulting peptides is fractionated by liquid chromatography (LC) into multiple, less complex, fractions, which are slowly but continuously injected by electrospray ionization into a tandem mass spectrometer. The fractions are then sequentially subjected to multiple cycles of MS/MS until the masses and sequences of many of the peptides have been determined and used to identify the proteins in the original biological sample through comparison with protein databases.
An example of the use of LC-MS/MS to identify many of the proteins in each organelle is seen in Figure 2. Cells from murine (mouse) liver tissue were mechanically broken to release the organelles, and the organelles were partially separated by density-gradient centrifugation. The locations of the organelles in the gradient were determined using immunoblotting with antibodies that recognized previously identified, organelle-specific proteins. Fractions from the gradient were then subjected to LC-MS/MS to identify the proteins in each fraction, and the distributions in the gradient of many individual proteins were compared with the distributions of the organelles. This strategy permitted the assignment of many individual proteins to one or more organelles (organelle proteome profiling). More recently, a combination of organelle purification, MS, biochemical localization, and computational methods has been used to show that at least a thousand distinct proteins are localized in the mitochondria of humans and mice.
Fig2. Density-gradient centrifugation and LC-MS/MS can be used to identify many of the proteins in organelles. (a) The cells in liver tissue were mechanically broken to release the organelles, and the organelles were partially separated by density-gradient centrifugation. The locations of the organelles—which were spread out through the gradient and somewhat overlapped with one another—were determined using immunoblotting with antibodies that recognized previously identified, organelle- specific proteins. Fractions from the gradient were subjected to proteolysis and LC-MS/MS to identify the peptides, and hence the proteins, in each fraction. Comparisons with the locations of the organelles in the gradient (called protein correlation profiling) permitted assignment of many individual proteins to one or more organelles (organelle proteome identification). (b) The hierarchical breakdown of data derived from the procedures in part (a). Note that not all proteins identified could be assigned to organelles and that some proteins were assigned to more than one organelle. [Data from L. J. Foster et al., 2006, Cell 125:187–199.]
Proteomics methods combined with molecular genetics methods are currently being used to identify all the protein complexes in eukaryotic cells. For example, in the yeast Saccharomyces cerevisiae, approximately 500 complexes, with an average of 4.9 distinct proteins per complex, have been identified. These complexes, in turn, are involved in at least 400 complex-to-complex interactions. Such systematic proteomic studies are providing new insights into the organization of proteins within cells and into how proteins work together to permit cells to live and function.
Phosphoproteomics, the identification and quantification of phosphorylation sites on the proteins in a complex mixture, is playing a growing role in the analysis of cell metabolism and regulation. As we have already learned, the reversible phosphorylation of proteins by kinases and phosphatases is a key mechanism for regulating proteins in cells. Phosphoproteomics permits the simultaneous determination of the phosphorylation states of many proteins and thus provides an important tool for analyzing complex cellular regulatory networks. Only a fraction—in some cases, only a small fraction—of a particular protein might be phosphorylated. Thus phosphoproteomic analysis can require 50–100 times more initial cell or tissue sample material (from about 2.5 to more than 20 mg of total cellular protein per sample) than does standard proteomic analysis. As a consequence, investigators usually use affinity chromatography methods with either metal-containing (e.g., Fe3+ or TiO2) or antibody-containing columns to separate phosphopeptides from nonphosphorylated peptides (phosphopeptide enrichment) prior to subjecting the phosphopeptides to LC-MS/ MS analysis.
