Developing Workflow for Simultaneous Analyses of Phosphopeptides and Glycopeptides

Kyung Cho Cho, Lijun Chen, Yingwei Hu, Michael Schnaubelt, Hui Zhang

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)58-66
Number of pages9
JournalACS chemical biology
Issue number1
StatePublished - Jan 18 2019

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine


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