Identification of protein expression at the whole proteome level includes quantitative measurement of proteins in a cell at a particular metabolic state. The classic protein separation methods involve two-dimensional gel electrophoresis followed by gel image analysis. Further identification includes finding of amino acid composition, peptide mass fingerprints, and sequences using mass spectrometry (MS). Finally, database searching is needed for protein expression identification.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a high-resolution technique that separates proteins by charge and mass. The gel is run in one direction in a pH gradient under a non-denaturing condition to separate proteins by isoelectric points (pI) and then in an orthogonal dimension under a denaturing condition to separate proteins by molecular weights (MW). This is followed by staining, usually silver staining, which is very sensitive, to show the position of all proteins. The result is a two-dimensional gel map; each spot on the map corresponds to a single protein being expressed. The stained gel can be further scanned and digitized for image analysis. However, not all proteins can be separated by this method or stained properly. One of the challenges of this technique is the separation of membrane proteins, which are largely hydrophobic and not readily solubilized. They tend to cluster in the aqueous medium of a two-dimensional gel. Subfractionation can be done to separate nuclear, cytosol, cytoskeletal, and other subcellular fractions to boost the concentrations of rare proteins and to reveal subcellular localizations of the proteins. Gel image analysis is the next step that helps to show differential global protein expression patterns. This analysis includes spot determination, quantitation, and normalization. Image analysis software is used to measure the center, edges, and densities of the spots. Comparing two-dimensional gel images from certain experiments can sometimes pose a challenge because the gels, unlike DNA microarrays, may shrink or warp. This requires the software programs to be able to stretch or maneuver one of the gels relative to the other to find a common geometry.
Once the proteins are separated on a two-dimensional gel, they can be further identified and characterized using Mass Spectrometry technique. In this procedure, the proteins from a two dimensional gel system are first digested in situ with a protease (e.g., trypsin). Protein spots of interest are excised from the two-dimensional gel. The proteolysis generates a unique pattern of peptide fragments of various MWs, which is termed a peptide fingerprint. The fragments can be analyzed with MS, a high-resolution technique for determining molecular masses. Currently, electrospray ionization MS and matrix-assisted laser desorption ionization (MALDI) MS are commonly used. These two approaches only differ in the ionization procedure used.
MS characterization of proteins is mostly dependent on Bioinformatics analysis. Once the peptide mass fingerprints or peptide sequences are determined, Bioinformatics programs can be used to search for the identity of a protein in a database of theoretically digested proteins. The purpose of the database search is to search exact or nearly exact matches.
ExPASY is a comprehensive proteomics web server with a suite of programs for searching peptide information from the SWISS-PROT and TrEMBL databases. ProFound and Mascot are also used for this purpose.