Improving Glycoproteomic Analysis Workflow by Systematic Evaluation of Glycopeptide Enrichment, Quantification, Mass Spectrometry Approach, and Data Analysis Strategies

Glycosylation is one of the most prevalent and crucial
protein
modifications. Quantitative site-specific characterization of glycosylation
usually requires sophisticated intact glycopeptide analysis using
glycoproteomics. Recent efforts have focused on the interrogation
of intact glycopeptide analyses using tandem mass spectrometry. However,
a systematic evaluation of the quantitative glycoproteomic workflow
is still lacking. This study compared different strategies for glycopeptide
enrichment alongside glycopeptide quantitation, as well as mass spectrometry
and data analysis strategies, providing a comprehensive assessment
of their efficacy. The ZIC-HILIC enrichment method demonstrated superior
performance, representing a 26% improvement in identified glycopeptiudes
compared to the MAX enrichment method. Quantification using TMT provided
high precision and throughput with an average CV of 8%. Through systematic
evaluation, this study established that the ZIC-HILIC enrichment method,
quantification with TMT, and collision energies of 25, 35, and 45
using tandem mass spectrometry are the optimal workflow for higher-energy
collisional dissociation (HCD) fragmentation, significantly enhancing
the analysis of intact glycopeptides. Precise energy adjustment is
crucial for the identification of certain glycans. Intact glycopeptides
were analyzed using different software tools to investigate the identification
and quantification of glycopeptides. By applying optimal settings,
5514 unique intact glycopeptides were in luminal and basal patient-derived
xenograft (PDX) characterized models, highlighting distinct glycosylation
profiles that may influence tumor behavior. This study offers a systematic
approach to evaluate glycoproteomic analysis workflow.