Developing Workflow for Simultaneous Analyses of Phosphopeptides and Glycopeptides

Abstract

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.

Optimal Dissociation Methods Differ for N- and O-glycopeptides

“…sceHCD has been shown to benefit reporter ion generation without detrimental effects to peptide identification in isobaric labeling experiments, 132 yet relatively few studies to date have employed isobaric labeling strategies for glycoproteomic experiments. 44,50,[133][134][135][136][137] Perhaps adoption of sceHCD methods for Nglycopeptides will enable more widespread use of isobaric labels, while combinations of HCD and EThcD would still permit isobaric label-based quantitation in O-glycoproteomic workflows. Alternatively, the benefits of data-independent acquisition (DIA), which largely relies on collisional dissociation, have been shown for N-glycoproteomics.…”

Advances in mass spectrometry‐based glycoproteomics: An update covering the period 2017–2021

“…As a result, a total of 1067 intact N-glycopeptides were identified in this study. Cho et al [115] combined the enrichment methods with the TMT labeling method for simultaneous analysis of phosphopeptides and glycopeptides from luminal and basal-like types of breast cancer patient-derived xenografts. This method allowed the quantification and identification of 17 582 phosphopeptides and 3468 glycopeptides.…”

Recent Advances in Mass Spectrometry-Based Glycomic and Glycoproteomic Studies of Pancreatic Diseases

“…Efforts to maximize the PTM information obtained with MS workflows have gained traction in recent years. Using LC-MS, glycosylation is often studied with phosphorylation, as they are among the most common PTMs (Glover et al, 2018;Cho et al, 2019;Cui et al, 2019;Zhou et al, 2020;Cui et al, 2021;Huang et al, 2021). Both PTMs are implicated in cell signaling, and O-glycans may modify Ser and Thr, also two possible residues for phosphorylation.…”