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Quantitative analysis of phosphorylation‐based protein signaling networks in the immune system by mass spectrometry

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Abstract Dynamic modification of cell proteins with phosphate is one of the key regulators of the cellular response to external stimuli. Phosphorylation‐based signaling networks mediate cell proliferation, differentiation, and migration, and their dysregulation is the basis of multiple diseases. However, the transient nature of the regulatory protein phosphorylation and low site occupancy mean that only a fraction of the protein is phosphorylated at a given time, and it is a challenge to measure the degree and dynamics of phosphorylation using traditional biochemical means. Technological advances in the field of mass spectrometry (MS) made it possible to generate large sets of phosphoproteomics data, probing the phosphoproteome with great depth, sensitivity, and accuracy. Therefore, quantitative phosphoproteomics emerged as one of the essential components of the systems biology approach for profiling of complex biological networks. Nowadays, the challenge lies in validation of the information and in its integration into the comprehensive models of cell decision processes. This article reviews the role of phosphoproteomics in systems biology, the MS‐based approach, and technical details of the methods. Recent examples of quantitative measurements and methodologies as well as applications to the studies of the immune system and infectious diseases are presented and discussed. WIREs Syst Biol Med 2011 3 368–376 DOI: 10.1002/wsbm.123 This article is categorized under: Laboratory Methods and Technologies > Proteomics Methods

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(a) Relative abundance of serine, threonine, and tyrosine phosphorylations. (b) Tyrosine kinase family, divided into receptor tyrosine kinase (RTK) and non‐receptor tyrosine kinase (non‐RTK) subfamilies, in relation to the total number of kinase sequences in the human genome.

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Example of the workflow for quantitative phosphorylation analysis using iTRAQ labeling. Cultured cells are stimulated with specific ligands for a series of time points and lysed without detergent. The lysates are treated with a reducing agent to break the disulfide bonds and the free cysteine residues are modified to prevent the reformation of disulfide bonds. Subsequently, the lysates are digested with trypsin, the samples are labeled with iTRAQ (up to eight conditions can be distinguished) and combined. For tyrosine phosphorylation analysis, immunoprecipitation (IP) with anti‐pTyr antibodies is performed prior to immobilized metal affinity chromatography (IMAC) enrichment; for global phosphorylation analysis, phosphopeptides are enriched only with IMAC. After elution from IMAC column, phosphorylated peptides are analyzed by LC‐MS/MS.

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