Pathway proteomics: global and focused approaches.

Biological pathways represent the relationships (reactions and interactions) between biological molecules in the context of normal cellular functions and disease mechanisms. Understanding the roles of proteins and signaling pathways expressed within disease, and their link to drug discovery and drug development are central in today's target-driven pharmaceutical processes. This article gives an overview of proteomics strategies, including global expression analysis as well as focused approaches using multidimensional separation by both gel- and liquid-phase techniques linked to mass spectrometry, as applied to two of the pathways involved in inflammatory diseases. In primary human cell studies, our group has annotated and identified thousands of proteins using both electrospray ionization and matrix-assisted laser desorption ionization (MALDI)-sequencing technology. Annotations made from gel images and chromatography fractionation, interfaced to high-end mass spectrometry sequence and structure identity, are cornerstones in cutting-edge protein expression profiling. Regarding phosphorylation mechanisms of kinases, the quantitative stoichiometry can be determined using affinity probe isolations. Another strategy involves micro-preparative sample processing, which has been used to analyze single-target phosphoproteins and their relative phospho-stoichiometry.