HiRIEF LC-MS allows for in depth quantitative analysis of the phosphoproteome.

In this project we aimed at improving phosphoproteomics analysis by taking advantage of high resolution isoelectric focusing (HiRIEF) fractionation. We developed a workflow that employs titanium dioxide phospho-enrichment, followed by isobaric labeling with Tandem Mass Tags (TMT) and HiRIEF on a broad pI range (immobilized pH gradient, IPG gel strips employed were 2.5-3.7 and 3-10, Phospho HiRIEF LC-MS). We analyzed HeLa cells untreated (four biological replicates), treated with pervanadate or arrested in mitosis (three biological replicates each). Employing a relatively low amount of material (300 μg of peptides), we identify 22,674 phosphorylation sites, of which 19,036 were localized with high confidence. We demonstrate isoelectric point dependent fractionation of the peptides based on the number of phosphate groups that they carry: 18% of the phospho-peptides identified with the IPG 2.5-3.7 gel strip are multiply phosphorylated peptides and they localize predominantly in the most acidic pI fractions. Identified phosphorylation sites include 1,198 tyrosine phosphorylation sites and 1,491 phospho-sites that were not previously reported in the PhosphoSitePlus database. Total protein quantification performed by standard HiRIEF on the same samples identified 9,185 proteins, of which 4,575 overlap with the proteins identified by Phospho HiRIEF LC-MS. Phosphorylation sites corresponding to these proteins were normalized to total protein abundance, resulting in 18,374 quantified phospho-sites. Kinase association analysis on the quantified phospho-sites resulted in identification of a subset that has putative functions during the mitotic phase and protein-protein interaction network analysis shows a high degree of connectivity of these putatively functional novel phospho-sites.