Phosphoproteomics in the Age of Rapid and Deep Proteome Profiling

Profiling Nicholas M. Riley†,‡ and Joshua J. Coon*,†,‡,§ †Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States ‡Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States ■ CONTENTS Sampling the Phosphoproteome 74 Generating Phosphopeptides 75 Enrichment Strategies 75 Chromatographic Separations 77 Mass Spectrometry Instrumentation 78 Data Acquisition Strategies 79 Balancing Throughput and Depth 79 Quantifying the Phosphoproteome 81 Stable Isotope Labeling 81 Label-Free Strategies 83 Confident Phosphosite Assignment 84 Tandem MS Approaches 84 Post-Acquisition Processing and Informatics 85 Biological Insights via Phosphoproteomics 87 PTM Cross-Talk 88 Phosphorylation beyond Serine, Threonine, and Tyrosine 88 Looking Forward 89 Author Information 89 Corresponding Author 89 Notes 89 Biographies 89 Acknowledgments 89 References 89