Global profiling of phosphopeptides by titania affinity enrichment.

Protein phosphorylation is a ubiquitous post-translational modification critical to many cellular processes. Large-scale unbiased characterization of phosphorylation status remains a major technical challenge in proteomics. In the present work, we evaluate and optimize titania-based affinity enrichment for global profiling of phosphopeptides from complex biological mixtures. We demonstrate that inclusion of glutamic acid in the sample loading buffer substantially reduced nonspecific binding of nonphosphorylated peptides to the titania while retaining the high binding affinity for phosphopeptides. The reduction in nonspecific peptide binding enhanced overall phosphopeptide recovery, ranging from 22 to 85%, and led to substantial improvement in large-scale global profiling. In addition, we observed that the overall identification of phosphopeptides was significantly enhanced by neutral loss-triggered MS (3) scans and respective use of multiple charge- and mass-dependent filtering criteria for MS (2) and MS (3) spectra. In conjunction with strong-cation exchange chromatography (SCX) for prefractionation, a total of 4002 distinct phosphopeptides were identified from SKBr3 breast cancer cells at false-positive rates of 3.7% and 5.5%, respectively, for singly and doubly phosphorylated peptides.