The rapid progress in gene sequencing has advanced another goal of biochemistry—elucidation of the proteome. The proteome is the complete set of proteins expressed and includes information about how they are modified, how they function, and how they interact with other molecules.

3.1 The Purification of Proteins Is an Essential First Step in Understanding Their Function

Proteins can be separated from one another and from other molecules on the basis of such characteristics as solubility, size, charge, and binding affinity. SDS–polyacrylamide gel electrophoresis separates the polypeptide chains of proteins under denaturing conditions largely according to mass. Proteins can also be separated electrophoretically on the basis of net charge by isoelectric focusing in a pH gradient. Ultracentrifugation and gel-filtration chromatography resolve proteins according to size, whereas ion-exchange chromatography separates them mainly on the basis of net charge. The high affinity of many proteins for specific chemical groups is exploited in affinity chromatography, in which proteins bind to columns containing beads bearing covalently linked substrates, inhibitors, or other specifically recognized groups. The mass of a protein can be determined by sedimentation-equilibrium measurements.

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3.2 Immunology Provides Important Techniques with Which to Investigate Proteins

Proteins can be detected and quantitated by highly specific antibodies; monoclonal antibodies are especially useful because they are homogeneous. Enzyme-linked immunosorbent assays and western blots of SDS– polyacrylamide gels are used extensively. Proteins can also be localized within cells by immunofluorescence microscopy and immunoelectron microscopy.

3.3 Mass Spectrometry Is a Powerful Technique for the Identification of Peptides and Proteins

Techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) allow the generation of ions of proteins and peptides in the gas phase. The mass of such protein ions can be determined with great accuracy and precision. Masses determined by these techniques act as protein name tags because the mass of a protein or peptide is precisely determined by its amino acid composition and, hence, by its sequence. In addition to chemical methods, such as the Edman degradation, tandem mass spectrometry enables the rapid and highly accurate sequencing of peptides. These sequences are rich in information concerning the kinship of proteins, their evolutionary relationships, and diseases produced by mutations. Knowledge of a sequence provides valuable clues to conformation and function. Mass spectrometric techniques are central to proteomics because they make it possible to analyze the constituents of large macromolecular assemblies or other collections of proteins.

3.4 Peptides Can Be Synthesized by Automated Solid-Phase Methods

Polypeptide chains can be synthesized by automated solid-phase methods in which the carboxyl end of the growing chain is linked to an insoluble support. The carboxyl group of the incoming amino acid is activated by dicyclohexylcarbodiimide and joined to the amino group of the growing chain. Synthetic peptides can serve as drugs and as antigens to stimulate the formation of specific antibodies. They can also be sources of insight into the relation between amino acid sequence and conformation.

3.5 Three-Dimensional Protein Structure Can Be Determined by X-ray Crystallography and NMR Spectroscopy

X-ray crystallography and nuclear magnetic resonance spectroscopy have greatly enriched our understanding of how proteins fold, recognize other molecules, and catalyze chemical reactions. X-ray crystallography is possible because electrons scatter x-rays. The diffraction pattern produced can be analyzed to reveal the arrangement of atoms in a protein. The three-dimensional structures of tens of thousands of proteins are now known in atomic detail. Nuclear magnetic resonance spectroscopy reveals the structure and dynamics of proteins in solution. The chemical shift of nuclei depends on their local environment. Furthermore, the spins of neighboring nuclei interact with each other in ways that provide definitive structural information. This information can be used to determine complete three-dimensional structures of proteins.

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