Development of Titania Particles Used for Phosphopeptide Enrichment in Mass Spectrometry-Based Phosphoproteomics

Abstract

Titanium dioxide, titania, is used as an a$nity support for enriching phosphopeptides in mass spectrometry- based phosphoproteomics. In this study, a variety of titania particles were prepared by calcination at di#erent temperatures or were obtained from di#erent manufacturers, and relationships between their physico-chemical properties and the selectivity in phosphopeptide enrichment was examined. Tryptic peptides from three phosphoproteins were employed for phosphopeptide enrichment by aliphatic hydroxyl acid-modified metal oxide chromatography (HAMMOC) using titania in the presence of lactic acid. The recovery of phosphopeptides increased with increasing calcination temperature, up to 500?, while the number of identified non- phosphopeptides decreased. For titania calcined at 800?, the retention of phoshopeptides as well as non- phosphopeptides was significantly reduced in the presence of lactic acid. Various physico-chemical parameters, such as crystalline form, pore size and surface area were examined for these calcined titania beads, as well as commercial titania beads. The findings indicate that the weight-loss from 130? to 800?, as determined by thermal analysis, was well correlated with the selectivity of phosphopeptides by HAMMOC. Based on these results, optimized titania beads were prepared and applied to 100 mg of a HeLa cell extract. More than 1,000 phosphorylation sites were successfully identified with a false positive rate of 1.4?, whereas 455 phosphoryla- tion sites were identified with a false positive rate of 4.1?when commercially available titania beads were used for HAMMOC