New separation tools for comprehensive studies of protein expression by mass spectrometry

Mass spectrometry has emerged as a core technique for protein identification and characterization because of its high sensitivity, accuracy, and speed of analysis. The most widespread strategy for studying global protein expression in biological systems employs analytical two-dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by enzymatic degradation of isolated protein spots, peptide mapping, and bioinformatics searches. Using this method, thousands of proteins can be resolved in a gel and their expression quantified. However, certain types of proteins possessing important cellular functions are not easily analyzed using this strategy. These proteins include membrane, low copy number, highly basic, and very large (> 150 kDa) and small (< 10 kDa) proteins. To meet the growing need to simultaneously monitor all types of proteins in a biological system, new separation strategies have emerged that are amenable to hyphenation to mass spectrometric techniques. This article will review these new techniques and examine their usefulness in studies of protein expression.