title: Targeted In-Depth Quantification of Signaling Using Label-Free Mass Spectrometry.
abstract: Protein phosphorylation encodes information on the activity of kinase-driven signaling pathways that regulate cell biology. This chapter discusses an approach, named TIQUAS (targeted in-depth quantification of signaling), to quantify cell signaling comprehensively and without bias. The workflow-based on mass spectrometry (MS) and computational science-consists of targeting the analysis of phosphopeptides previously identified by shotgun liquid chromatography tandem MS (LC-MS/MS) across the samples that are being compared. TIQUAS therefore takes advantage of concepts derived from both targeted (data-independent) and data-dependent acquisition methods; phosphorylation sites are quantified in all experimental samples regardless of whether or not these phosphopeptides were identified by MS/MS in all runs. As a result, datasets are obtained containing quantitative information on several thousand phosphorylation sites in as many samples and replicates as required in the experimental design, and these rich datasets are devoid of a significant number of missing data points. This chapter discussed the biochemical, analytical, and computational procedures required to apply the approach and for obtaining a biological interpretation of the data in the context of our understanding of cell signaling regulation and kinase-substrate relationships.
authors.affiliation: Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, London, United Kingdom. Electronic address: p.cutillas@qmul.ac.uk.
authors.forename: P R
authors.initials: PR
authors.lastname: Cutillas
mesh_terms: D013058:Mass Spectrometry / Q000379:methods*; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides
keywords: Cell signaling; Inhibitor; Kinase; Mass spectrometry; Phosphoproteomics; Phosphorylation; Quantification; Systems biology