Multidimensional separations for protein/peptide analysis in the post-genomic era

Abstract

Proteomics is the study of all or part of the protein complement of genes in an organism, often involving the analysis of complex protein/peptide samples. Such complex samples are beyond the separation capacity of 1-D separation techniques. This review describes several multidimensional separations for proteins and peptides. First, several variants of 2-D liquid chromatography (2DLC) are reviewed, including coupled size exclusion-reversed phase, ion exchange-reversed phase, and reversed phase-reversed phase chromatography. Second, we describe coupled liquid chromatography and capillary electrophoresis methods. Finally, a multidimensional protein identification technique (MudPIT) is explained in detail. Each of the described techniques has a much higher separation capacity than 1-D methods and can potentially be automated for high-throughput experiments. In particular, MudPIT takes advantage of both the high separation capacity of 2DLC and the powerful peptide characterization ability of tandem mass spectrometry to analyze complex protein samples. Additional applications and developments of multidimensional liquid separations for proteomics are expected in the future.