A novel titanium dioxide-polydimethylsiloxane plate for phosphopeptide enrichment and mass spectrometry analysis

Abstract(#br)The phosphorylation of proteins is a major post-translational modification that is required for the regulation of many cellular processes and activities. Mass spectrometry signals of low-abundance phosphorylated peptides are commonly suppressed by the presence of abundant non-phosphorylated peptides. Therefore, one of the major challenges in the detection of low-abundance phosphopeptides is their enrichment from complex peptide mixtures. Titanium dioxide (TiO 2 ) has been proven to be a highly efficient approach for phosphopeptide enrichment and is widely applied. In this study, a novel TiO 2 plate was developed by coating TiO 2 particles onto polydimethylsiloxane (PDMS)-coated MALDI plates, glass, or plastic substrates. The TiO 2 -PDMS plate (TP plate) could be used for on-target MALDI-TOF analysis, or as a purification plate on which phosphopeptides were eluted out and subjected to MALDI-TOF or nanoLC-MS/MS analysis. The detection limit of the TP plate was ∼10-folds lower than that of a TiO 2 -packed tip approach. The capacity of the ∼2.5\u003cce:hsp sp=\"0.25\"/\u003emm diameter TiO 2 spots was estimated to be ∼10\u003cce:hsp sp=\"0.25\"/\u003eμg of β-casein. Following TiO 2 plate enrichment of SCC4 cell lysate digests and nanoLC-MS/MS analysis, ∼82% of the detected proteins were phosphorylated, illustrating the sensitivity and effectiveness of the TP plate for phosphoproteomic study.