Cancer Signaling Network Analysis by Quantitative Mass Spectrometry

Abstract

Global analysis of protein phosphorylation by mass spectrometry (MS) provides unbiased, discovery-based, site-specific monitoring of phosphorylation sites governing cell signaling networks involved in cancer progression and therapeutic resistance. In this chapter, advances in MS instrumentation and methodology for the identification and quantification of protein phosphorylation are discussed. These topics include (1) advantages and limitations of current MS-based protocols, (2) fundamentals of phosphopeptide fragmentation and identification, (3) selection of MS instrumentation, (4) fractionation and enrichment methods for detecting phosphorylated proteins/peptides, and (5) methods for phosphoproteome quantification. The final two topics represent the most important subjects of this chapter, as fractionation, enrichment, and quantification are crucially important to the generation of high quality MS-based phosphoproteomic data. These sections detail the use of immunoaffinity enrichment, immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and strong cation exchange (SCX) chromatography as key methods for enriching and fractionating complex biological samples for phosphoproteomic analysis. Quantification of changes in signaling networks at the phosphoproteomic level through metabolic labeling (e.g., SILAC), chemical modification (e.g., iTRAQ), or label-free quantification is presented. As significant progress in detection and quantification strategies in phosphoproteomic research has arisen over the last decade, implementation of these approaches will enhance our understanding of cell signaling networks involved in cancer progression and thereby improve therapeutic targeting and monitoring of therapeutic efficacy.KeywordsMass spectrometryPosttranslational modificationProteomicsPhosphoproteomicsGlobal analysisSpectrumPeptideMatrix-assisted laser desorption ionizationElectrospray ionizationHigh-performance liquid chromatographyFractionationEnrichmentCollision-activated dissociationIon trapElectron capture dissociationElectron transfer dissociationValidationQuadrupole time-of-flightSILACiTRAQMultiple reaction monitoringAmino acid residueImmunoaffinity purificationImmobilized metal affinitychromatographyMetal oxide affinity chromatographyStrong cation exchange chromatographyLabel-free quantificationMetabolic labelingStable isotopeChemical modificationSpectral counting