Figure 1. Comaparison of elution buffers for phosphopeptide analysis using TiO₂.

One pmol of a tryptic digest of α- and β-casein was loaded onto TiO₂ using an automated multidimensional liquid chromatography system (MDLC). Bound peptides were washed, eluted onto a C18 trap column and subjected to online LC-MS/MS analysis, peptide-spectral matches were made using the SEQUEST algorithm. Four different elution buffers were tested. Spectra count results of the four most abundant phosphopeptides, and the sum of all spectra counts from all identified phosphopeptides are presented. Spectra count results are normalized to those observed for the 200 mM NH₄HCO₃ pH 9 elution buffer. Non-normalized data of all identified phosphopeptides is included in Supplemental Information (S-Table 7). The loading and wash buffers contained 10% acetonitrile and 1% formic acid.