Proteomics technologies for the global identification and quantification of proteins

Abstract

This review provides an introduction for the nonspecialist to proteomics and in particular the major approaches available for global protein identification and quantification. Proteomics technologies offer considerable opportunities for improved biological understanding and biomarker discovery. The central platform for proteomics is tandem mass spectrometry (MS) but a number of other technologies, resources, and expertise are absolutely required to perform meaningful experiments. These include protein separation science (and protein biochemistry in general), genomics, and bioinformatics. There are a range of workflows available for protein (or peptide) separation prior to tandem MS and subsequent bioinformatics analysis to achieve protein identifications. The predominant approaches are 2D electrophoresis (2DE) and subsequent MS, liquid chromatography-MS (LC-MS), and GeLC-MS. Beyond protein identification, there are a number of well-established options available for protein quantification. Difference gel electrophoresis (DIGE) following 2DE is one option but MS-based methods (most commonly iTRAQ-Isobaric Tags for Relative and Absolute Quantification or SILAC-Stable Isotope Labeling by Amino Acids) are now the preferred options. Sample preparation is critical to performing good experiments and subcellular fractionation can additionally provide protein localization information compared with whole cell lysates. Differential detergent solubilization is another valid option. With biological fluids, it is possible to remove the most abundant proteins by immunodepletion. Sample enrichment is also used extensively in certain analyses and most commonly in phosphoproteomics with the initial purification of phosphopeptides. Proteomics produces considerable datasets and resources to facilitate the necessary extended analysis of this data are improving all the time. Beyond the opportunities afforded by proteomics there are definite challenges to achieving full proteomic coverage. Proteomes are highly complex and identifying and quantifying low abundance proteins is a significant issue. Additionally, the analysis of poorly soluble proteins, such as membrane proteins and multiprotein complexes, is difficult. However, it is without doubt that proteomics has already provided significant insights into biological function and this will continue as the technology continues to improve. We also anticipate that the promise of proteomics in terms of biomarker discovery will increasingly be realized.