Biomarker discovery: proteome fractionation and separation in biological samples

Proteomics, analogous with genomics, is the analysis of the protein complement present in a cell, organ, or organism at any given time. While the genome provides information about the theoretical status of the cellular proteins, the proteome describes the actual content, which ultimately determines the phenotype. The broad application of proteomic technologies in basic science and clinical medicine has the potential to accelerate our understanding of the molecular mechanisms underlying disease and may facilitate the discovery of new drug targets and diagnostic disease markers. Proteomics is a rapidly developing and changing scientific discipline, and the last 5 yr have seen major advances in the underlying techniques as well as expansion into new applications. Core technologies for the separation of proteins and/or peptides are one- and two-dimensional gel electrophoresis and one- and two-dimensional liquid chromatography, and these are coupled almost exclusively with mass spectrometry. Proteomic studies have shown that the most effective analysis of even simple biological samples requires subfractionation and/or enrichment before protein identification by mass spectrometry. Selection of the appropriate technology or combination of technologies to match the biological questions is essential for maximum coverage of the selected subproteome and to ensure both the full interpretation and the downstream utility of the data. In this review, we describe the current technologies for proteome fractionation and separation of biological samples, based on our lab workflow for biomarker discovery and validation.