Site-specific degree of phosphorylation in proteins measured by liquid chromatography-electrospray mass spectrometry.

This review focuses on quantitative protein phosphorylation analysis based on coverage of both the phosphorylated and nonphosphorylated forms. In this way, site-specific data on the degree of phosphorylation can be measured, generating the most detailed level of phosphorylation status analysis of proteins. To highlight the experimental challenges in this type of quantitative protein phosphorylation analysis, we discuss the typical workflows for mass spectrometry-based proteomics with a focus on the quantitative analysis of peptide/phosphopeptide ratios. We review workflows for measuring site-specific degrees of phosphorylation including the label-free approach, differential stable isotope labeling of analytes, and methods based on the addition of stable isotope labeled peptide/phosphopeptide pairs as internal standards. The discussion also includes the determination of phosphopeptide isoform abundance data for multiply phosphorylated motifs that contain information about the connectivity of phosphorylation events. The review closes with a prospective on the use of intact stable isotope labeled proteins as internal standards and a summarizing discussion of the typical accuracies of the individual methods.