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Phosphorylation is an important regulator of cell function in eukaryotes. This post-translational modification can alter protein localization, regulate protein function, and stabilize and mediate their interactions. Due to their associated negative charge, phosphopeptides are often poorly ionized compared to their non-phosphorylated counterparts, and their analysis is often complicated due to their low cellular abundance. Therefore, it is critical to selectively enrich the phosphopeptides prior to MS analysis.
In this study, we have evaluated a new way of enriching phosphopeptides by using a metal oxide–based solid-phase extraction (SPE) microscale device where the eluent is analyzed by LC MALDI MS. This sorbent has a high affinity for phosphopeptides, and the problem of acidic peptide adsorption associated with immobilized metal-ion affinity chromatography, the previous technique, is greatly minimized.
Furthermore, recent developments in MALDI and LC-MALDI spotting devices allow the coupling of the off-line chromatographic separation step to the subsequent MS analysis.
Data will be presented showing the enrichment of phosphopeptides from a digest of a single phosphoprotein, beta casein, analyzed by LC-MALDI MS, where enhanced performance was observed in terms of the selectivity of the phosphopeptides. Further analysis of more complex protein mixtures (six and twelve standard proteins) which contain phosphopeptides will be presented, demonstrating the selectivity achieved using an aromatic carboxylic acid additive.