Selective Enrichment of Phosphopeptides on MALDI PlatesThe intracellular phosphorylation of serine, threonine, or tyrosine residues of proteins provides a mechanism for the cell to switch on or off many diverse processes, such as kinase cascade activation and gene transcription. Hence, a profile of protein phosphorylation is vital for understanding cell regulation. Currently, MS is the most powerful technique for studying protein post-translational modifications, but profiling of phosphorylation typically requires enrichment of phosphopeptides after proteolytic digestion because of the low stoichiometry of protein phosphorylation. One strategy to minimize sample consumption and simplify enrichment is capture of the desired species directly on the matrix assisted laser desorption/ionization (MALDI) plate. We recently developed a method for growing polymer brushes on MALDI plates and modifying them with titania clusters (Figure a below) that selectively bind phosphopeptides prior to rinsing, elution, and MALDI-MS (Figure b below).
(a) Schematic drawing of the formation of the polymer-oxoTi hybrid structure, and the binding and elution of phosphopeptides in this material. (b) Procedures for on-plate phosphopeptide enrichment using Au-P-oxoTi.
Enrichment of phosphoprotein digests lead to mass spectra dominated with signals from phosphopeptides. In a recent paper, J. Proteome Res. 9, 3005–3015 (2010), we used these modified MALDI plates to identify phosphoproteins that are associated with p65, an ubiquitous protein that is partially responsible for regulating inflammation as well as the life cycle of lymphoid cells during the immune response. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.