The 2-DE gel-based methods in proteomics. (A) Most techniques currently used in proteomics involve the separation of the vast number of proteins present in a cell or tissue at a given time prior to analysis by MS and recognition and characterization using bioinformatics techniques. The protein separation can be performed at the protein or peptide level. One widespread methodology is 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE), which separates proteins according to their isoelectric points and molecular weights. One advantage of this approach is the ability to separate differentially posttranslationally modified forms of the same protein. A key disadvantage is that complete coverage of the proteome cannot be achieved, because some proteins will not enter the gel. Relative quantification of proteins between samples was in the past achieved by intergel comparisons following staining (Table 1). (B) The recently deployed technique of fluorescence 2-dimensional difference gel electrophoresis (2-D DIGE) for the relative quantification of proteins from up to 3 cell states on the same gel offers a greater reproducibility than previous approaches. Prior to the 2-D SDS-PAGE separation, the samples are covalently labeled with succinimidyl esters of different cyanide dyes (Cy2, Cy3, and Cy5). The N-hydroxy-succinimidyl esters undergo nucleophilic substitution reaction with the lysine ϵ amine groups to give an amide. The samples are then mixed together and separated on the same 2-D SDS-PAGE gel. The gel is scanned at the different excitation frequencies, the images merged, and the difference in protein abundance calculated. However, to maintain the solubility of the proteins during the electrophoretic separation, 2-D DIGE12 requires that only 1% to 2% of the lysine residues be derivatized (the derivatization increases the hydrophobicity of the proteins). Recently, a new set of dyes intended to fluorescently label all cysteine residues within a protein have been made available and these offer greater sensitivity.91 (C) Following separation, the protein spot is usually excised, subjected to in-gel enzymatic proteolysis, and analyzed by MS, usually MALDI-MS. The MALDI peptide fingerprint is often sufficient for confident protein recognition. For further confirmation, amino acid sequence information, or recognition of low-abundance proteins, tandem mass spectrometry (MS/MS) analysis is performed, generally using electrospray (ES) ionization. A prevalent method that separates proteins at the peptide level is liquid chromatography (LC), usually used in conjunction with MS/MS (LC-MS/MS).
