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Glycopeptide Capture for Cell Surface Proteomics
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Optimal Dissociation Methods Differ for N- and O-Glycopeptides.

Nicholas M Riley1, Stacy A Malaker1, Marc D Driessen1

  • 1Department of Chemistry, Stanford University, Stanford, California 94305-6104, United States.

Journal of Proteome Research
|June 6, 2020
PubMed
Summary

Comparing mass spectrometry methods for glycopeptide analysis reveals distinct performance for N- and O-glycans. Electron transfer dissociation (ETD) based methods are crucial for O-glycopeptides, while higher-energy collisional dissociation (HCD) suffices for N-glycopeptides.

Keywords:
ETDEThcDN-glycopeptidesO-glycopeptideselectron transfer dissociationfragmentationglycoproteomicssceHCDstepped collision energy high-energy collisional dissociationtandem MS

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Area of Science:

  • Proteomics and Mass Spectrometry
  • Glycoscience
  • Biochemistry

Background:

  • Site-specific glycosylation analysis is vital for understanding glycopeptide function.
  • Tandem mass spectrometry (MS/MS) is key for glycopeptide interrogation.
  • Higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) are common MS/MS activation methods.

Purpose of the Study:

  • To systematically compare the performance of HCD, stepped collision energy HCD (sceHCD), ETD, and electron transfer dissociation with HCD supplemental activation (EThcD) for intact glycopeptide analysis.
  • To determine the suitability of these methods for both N-linked (N-) and O-linked (O-) glycoproteomics.
  • To guide the selection of appropriate MS/MS methods for different glycopeptide types.

Main Methods:

  • Intact glycopeptide analysis using four MS/MS methods: HCD, sceHCD, ETD, and EThcD.
  • Comparative analysis of spectral quality and number of identifications for N-glycopeptides and O-glycopeptides.
  • Systematic evaluation of method performance across different glycopeptide classes.

Main Results:

  • For N-glycopeptides, HCD and sceHCD yielded similar identification numbers, with sceHCD providing better spectral quality. ETD-based methods were not superior for routine N-glycoproteomics.
  • For O-glycopeptides, ETD-based methods, particularly EThcD, were indispensable for site-specific characterization.
  • HCD-centric methods were insufficient for robust O-glycopeptide analysis, unlike their utility for N-glycopeptides.

Conclusions:

  • The choice of MS/MS method is critical and depends on whether analyzing N- or O-glycopeptides.
  • EThcD is essential for comprehensive O-glycopeptide site-specific characterization.
  • Future glycoproteomic strategies must be tailored to the specific requirements of O-glycopeptide analysis.