Catch me if you can: Mass spectrometry‐based phosphoproteomics and quantification strategies

Abstract Phosphorylation of proteins is one of the most prominent PTMs and for instance a key regulator of signal transduction. In order to improve our understanding of cellular phosphorylation events, considerable effort has been devoted to improving the analysis of phosphorylation by MS‐based proteomics. Different enrichment strategies for phosphorylated peptides/proteins, such as immunoaffinity chromatography (IMAC) or titanium dioxide, have been established and constantly optimized for subsequent MS analysis. Concurrently, specific MS techniques were developed for more confident identification and phosphorylation site localization. In addition, more attention is paid to the LC‐MS instrumentation to avoid premature loss of phosphorylated peptides within the analytical system. Despite major advances in all of these fields, the analysis of phosphopeptides still remains far from being routine in proteomics. However, to reveal cellular regulation by phosphorylation events, not only qualitative information about the phosphorylation status of proteins but also, in particular, quantitative information about distinct changes in phosphorylation patterns upon specific stimulation is mandatory. Thus, yielded insights are of outstanding importance for the emerging field of systems biology. In this review, we will give an insight into the historical development of phosphoproteome analysis and discuss its recent progress particularly regarding phosphopeptide quantification and assessment of phosphorylation stoichiometry.