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

摘要

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 (TiO2) has been proven to be a highly efficient approach for phosphopeptide enrichment and is widely applied. In this study, a novel TiO2 plate was developed by coating TiO2 particles onto polydimethylsiloxane (PDMS)-coated MALDI plates, glass, or plastic substrates. The TiO2-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 TiO2-packed tip approach. The capacity of the ～2.5 mm diameter TiO2 spots was estimated to be ～10 μg of β-casein. Following TiO2 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.