Chapter 13 Analysis of Protein-Tyrosine Phosphorylation by Mass Spectrometry

Abstract

Publisher Summary This chapter provides an overview of mass spectrometry (MS)-based techniques and strategies for the analysis of tyrosine phosphorylation. The analysis of serine and threonine phosphorylation is reviewed as well. Analysis of tyrosine phosphorylation has been challenging for four reasons: (1) many signaling proteins that can be tyrosine phosphorylated are present at low abundance within the cell, (2) the stoichiometry of tyrosine-phosphorylated proteins is often very low, (3) proteins often have multiple phosphorylation sites and are heterogeneously phosphorylated, and (4) tyrosine phosphorylation is highly dynamic. Several analytical techniques are available for the analysis of phosphorylation, including Edman sequencing, 32 P labeling, and mass spectrometry (MS). Compared to other methods, MS offers the advantages of high sensitivity, high speed, ability to localize the phosphorylation sites and suitability for large-scale analysis. While top-down approaches for the analysis of phosphotyrosine (pTyr) are not yet widely used—as high resolution MS instrumentation and the appropriate software for data analysis improve and become easier to use—this powerful approach has the capability to become much more popular due to its ability to achieve complete protein sequence coverage.