Protein phosphorylation controls many basic cellular processes, such as cell growth, differentiation, migration, metabolism, and cell death, and its study can provide key insights into the signal transduction pathways that are activated in cells in response to different stimuli, such as growth factor stimulation or exposure to toxicants. In Phospho-Proteomics: Methods and Protocols, expert researchers contribute both well-established protocols and some of the newest strategies for the identification and evaluation of protein phosphorylation on Tyr, Ser, and Thr residues, including topics such as 2-dimensional gel electrophoresis and protein phosphorylation, enrichment of phospho-proteins and peptides, quantitative analysis of phosphorylation by labeling and MS analysis, and antibody and kinase arrays. Written in the highly successful Methods in Molecular Biology series format, chapters include brief introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Phospho-Proteomics: Methods and Protocols is an ideal reference for both new and experienced scientists who wish to gain insight into the current developments in the field and to find inspiration to pursue its future.

Part I: Protein Phosphorylation and 2D Gel Electrophoresis A High-Resolution Two Dimensional Gel- and Pro-Q DPS-Based Proteomics Workflow for Phosphoprotein Identification and Quantitative Profiling. Ganesh K. Agrawal and Jay J. Thelen Differential Phospho-Protein Labeling (DIPPL) using 32P and 33P Aviva M. Tolkovsky and Andreas Wyttenbach Part II: Enrichment of Phospho-Proteins and Peptides Identification of Oxidative Stress-Induced Tyrosine Phosphorylated Proteins by Immunoprecipitation and Mass Spectrometry Ma Carmen Duran, Hong-Lin Chan and John F. Timms Enrichment and Characterization of Phosphopeptides by Immobilized Metal Affinity Chromatography (IMAC) and Mass Spectrometry Tine E. Thingholm and Ole N. Jensen The Use of Titanium Dioxide Micro-Columns to Selectively Isolate Phosphopeptides from Proteolytic Digests Tine E. Thingholm and Martin R. Larsen Enrichment and Separation of Mono- and Multi-Phosphorylated Peptides Using Sequential Elution from IMAC prior to Mass Spectrometric Analysis Tine E. Thingholm, Ole N. Jensen, and Martin R. Larsen Use of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) for Phosphotyrosine Protein Identification and Quantitation Guoan Zhang and Thomas A. Neubert Hydrophilic Interaction Chromatography for Fractionation and Enrichment of the Phosphoproteome Dean E. McNulty and Roland S. Annan SILAC for Global Phosphoproteomic Analysis Genaro Pimienta and Akhilesh Pandey Quantitative Phosphoproteomics Based on Soluble Nanopolymers Anton Iliuk and W. Andy Tao Profiling the Tyrosine Phosphorylation State Using SH2 domains Kevin Dierck, Kazuya Machida, Bruce J. Mayer, and Peter Nollau Part III: Phospho-Protein Analysis by Mass Spectrometry An Overview of the Qualitative Analysis of Phosphoproteins by Mass Spectrometry Philip R. Gafken The Analysis of Phosphoproteomes by Selective Labeling and Advanced Mass Spectrometric Techniques Angela Amoresano, Claudia Cirulli, Gianluca Monti, Eric Quemeneur and Gennaro Marino On-line Liquid Chromatography Electron Capture Dissociation for the Characterisation of Phosphorylation Sites in Proteins Steve M. Sweet and Helen J. Cooper Quantification of Protein Phosphorylation by µLC- ICP-MS Ralf Krüger, Nico Zinn, and Wolf D. Lehmann Reverse-Phase Diagonal Chromatography for Phosphoproteome Research Kris Gevaert and Joel Vandekerckhove Chemical Tagging Strategies for Mass Spectrometry-based Phosphoproteomics Alexander Leitner and Wolfgang Lindner Part IV: Arrays to Study Protein-Phosphorylation Antibody Array Platform to Monitor Protein Tyrosine Phosphorylation in Mammalian Cells. Alicia S. Chung and Y. Eugene Chin Protein Tyrosine Kinase Characterization Based on Fully Automated Synthesis of (phospho)Peptide arrays in Microplates W. Carl Saxinger Kinome Profiling Using Peptide Arrays in Eukaryotic Cells. Kaushal Parikh, Maikel P. Peppelenbosch, and Tita Ritsema Part V: Bioinformatics ProMoST: A Tool for Calculating the pI and Molecular Mass of Phosphorylated and Modified Proteins on 2 Dimensional Gels Brian D. Halligan Kinase Specific Prediction of Protein Phosphorylation Sites Martin L. Miller and Nikolaj Blom Reconstructing Regulatory Kinase Pathways from Phosphopeptide Data: A Bioinformatics Approach Lawrence G. Puente, Robin E.C. Lee, and Lynn A. Megeney