Developing Workflow for Simultaneous Analyses of Phosphopeptides and Glycopeptides

Enrichment of modified peptides from
global peptides is inevitable
in mass spectrometric analysis protein modifications because of their
importance in the study of cellular functions and low abundance in
the global proteomic analysis. Recent advances in enrichment methods
for modified peptides such as phosphopeptides and intact glycopeptides
(IGPs) show that the methods for proteomic analyses of both protein
modifications are robust. We have recently observed and reported a
large number of IGPs from phosphoproteomic analysis using IMAC-based
phosphopeptides enrichment procedure. To determine whether phosphorylated
peptides could be specifically isolated from coenriched IGPs in IMAC
experiments with different pH, IMAC procedures were performed at different
pH conditions, and we found that the enrichment of phosphopeptides
at pH 2.0 was the optimal condition for having the highest number
of phosphopeptide identifications; however, coenrichment of phosphopeptides
and glycopeptides was inevitable in the entire pH range. The hydrophilic
enrichments of IGPs performed before or after IMAC
enrichment were evaluated subsequently to determine the optimal workflow
for simultaneous analyses of phosphopeptides and glycopeptides, and
IMAC enrichment followed by hydrophilic enrichment was chosen as the
optimized workflow. Applying the workflow to the TMT-labeled peptides
from luminal and basal-like type of breast cancer patient-derived
xenograft (PDX) models allowed quantitative analyses of phospho- and
glycoproteomics with 17582 phosphopeptides and 3468 glycopeptides
identified, and 1237 phosphopeptides and 236 glycopeptides showed
significant expression differences between luminal and basal-like,
respectively. This method allows simultaneous analyses of phosphoprotein
and glycoprotein modifications, extending our understanding of roles
of glycosylation and phosphorylation in biology and diseases.