High-Performance Separations and Mass Spectrometric Methods for High-Throughput Proteomics using accurate Mass Tags

Abstract

The aim of strategy for proteome analysis is to exploit a combination of instrumental and methodological approaches to provide broad proteome coverage, high sensitivity, and the capability for greatly increased throughput compared with conventional technologies. The chapter reviews the technological basis and progress toward a global proteomics strategy that aims to provide large improvements in sensitivity, dynamic range, comprehensiveness, and throughput based on the use of polypeptide “accurate mass tags” (AMTs). The two-stage strategy exploits a single high-resolution capillary liquid chromatography (LC) separation combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate polypeptide AMTs for a specific organism, tissue, or cell type. The key attractions of the approach include the feasibility of completely automated high-confidence protein identification, extensive proteome coverage, and the capability for exploiting stable isotope-labeling methods for high-precision abundance measurements. Additional developments—including the use of multiplexed MS/MS capabilities and methods for dynamic range expansion of proteome measurements—are also described that promise to further extend the quality of measurements and their extension to much more challenging mammalian proteomes. Although increases in sensitivity can be advanced by different means, dynamic range expansion is a key factor when dealing with complex mixtures in which several species may be detected in a single spectrum.