Phosphoproteomics using CZE-MS/MS

ABSTRACT: Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide dataset is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Preliminary investigations demonstrated that predicted and observed electrophoretic mobility of phosphopeptides containing one phosphoryl group had good linear correlations (R2≥0.94). Adding one phosphoryl group on a peptide decreased its electrophoretic mobility dramatically because the phosphoryl group reduced the peptide’s charge by roughly 1 based on our experimental data. Phosphopeptides tended to migrate significantly slower than unphosphopeptides in the CZE separation capillary and phosphorylated and unphosphorylated forms of peptides were separated well by CZE. The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Liangliang Sun  

LAB HEAD: Liangliang Sun

PROVIDER: PXD012255 | Pride | 2019-01-16

REPOSITORIES: Pride