Identifying where these modifications occur and what part they play in cellular processes is key to gaining a greater understanding of various disease mechanisms as well as our ability to design drugs that prevent disease onset. The latest research, published in an early access article in the Journal of Proteome Research, describes the use of a titania-based affinity enrichment step to aid in the identification of phosphorylated peptides Protein post-translational modifications (PTMs) play a critical role in cell signalling pathways, regulating various cellular processes by changing either the structural or chemical environment of the proteins. This regulation of intercellular communication, cell growth, proliferation, differentiation and apoptosis can be achieved via a variety of reversible modifications including glycosylation or phosphorylation. "While a variety of mass-spectrometry (MS)-based proteomic techniques have been established for the identification and relative quantification of proteins in complex biological mixtures, large-scale analysis of phosphorylation at the proteome-wide scale remains a substantial challenge," write the authors. The use of titania (TiO2) to selectively adsorb negatively charged phosphopeptides has been previously described, however the author's believe these approaches have met with varying degrees of success and have been "greatly compromised by the detection of vast amounts of non-phosphorylated peptides that are retained". In this latest study, the researchers show that the inclusion of glutamic acid in the sample loading buffer significantly reduces the adsorption of non-phosphorylated peptides to titania beads while retaining the beads high binding affinity for phosphopeptides. After the non-phosphorylated peptides had been washed from the beads, the phosphopeptides were eluted by washing with a solution of ammonium hydroxide in acetonitrile. The enriched samples were then analysed using LC-MS (liquid chromatography - mass spectrometry) techniques on a Thermo Scientific LTQ-MS. The researchers used various MSn techniques to fragment and detect the peptides before searching human protein sequence databases to identify the fragments. Using this methodology they identified a total of 4002 non-redundant phosphopeptides from lysates of human SKBr3 cells. In comparison, the researchers only identified 1298 phosphopeptides using an identical protocol involving phthalic acid instead of glutamic acid.