Sample preparation and analytical strategies for large-scale phosphoproteomics experiments

Reversible protein phosphorylation is an important post-translational modification that controls a wide range of protein functions including enzyme activity, subcellular localisation, protein degradation, intra- and inter-molecular protein interactions. Significant advances in both phosphopeptide enrichment methods and sensitive mass spectrometry instrumentation have been achieved over the past decade to facilitate the large-scale identification of protein phosphorylation in humans and different animal and microbial model systems. While mass spectrometry provides the ability to identify thousands of phosphorylation sites in a single experiment, the further understanding of the functional significance of this modification on protein substrates requires detailed information on the changes in phosphorylation stoichiometry and protein abundance across experimental paradigms. This review presents different sample preparation methods and analytical strategies used in mass spectrometry-based phosphoproteomics to profile protein phosphorylation and unravel the regulation of this modification on protein function.

► Different enrichment strategies provide the ability to profile and identify thousands of phosphosites from complex cell extracts. ► Quantitative phosphoproteomics determines relative changes in protein phosphorylation and site occupancy. ► Comprehensive phosphoproteomics strategies can be used to assess site-specific variation of stoichiometry that affects protein function and regulation. ► Information on site stoichiometry and conservation can be used to prioritize phosphorylation sites for subsequent functional studies.