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Phosphorylation is one of the most common of the reversible post-translational modifications, but also one of the most challenging. During the last few years, phosphopeptide analysis has benefited from the rapid improvement in biological mass spectrometry, but also from innovations such as immobilized metal affinity chromatography, the development of antibodies specific for phosphorylated amino acids, and most recently, chromatography using TiO2.
During our analysis of the saliva phosphoproteome, we were concerned that there is a bias against monophosphorylated peptides when using TiO2 columns. We therefore decided to explore alternatives that rely on fractionation rather than affinity purification. Using the approach of Roepstorff and colleagues of packing columns in pipette tips to make disposable microcolumns, and combining strong cationic exchange (SCX) together with hydrophilic interaction liquid chromatography (HILIC), we have been able to fractionate peptides from digests of total saliva, and isolate phosphopeptides in a few fractions with a minimum of non-phosphorylated peptides. In preliminary experiments using about 6 μg of saliva, we have been able to map out phosphorylation sites from proteins such as the salivary acid proline-rich phosphoprotein 1/2, the salivary α-amylase, and statherin. A comparison of isolation of phosphopeptides using TiO2 vs. a combination of SCX and HILIC will be presented.