Comparison of Collisional and Electron-Based Dissociation Modes for Middle-Down Analysis of Multiply Glycosylated Peptides

  • Kshitij Khatri
  • Yi Pu
  • Joshua A. Klein
  • Juan Wei
  • Catherine E. Costello
  • Cheng Lin
  • Joseph Zaia
Focus: Mass Spectrometry in Glycobiology and Related Fields: Research Article


Analysis of singly glycosylated peptides has evolved to a point where large-scale LC-MS analyses can be performed at almost the same scale as proteomics experiments. While collisionally activated dissociation (CAD) remains the mainstay of bottom-up analyses, it performs poorly for the middle-down analysis of multiply glycosylated peptides. With improvements in instrumentation, electron-activated dissociation (ExD) modes are becoming increasingly prevalent for proteomics experiments and for the analysis of fragile modifications such as glycosylation. While these methods have been applied for glycopeptide analysis in isolated studies, an organized effort to compare their efficiencies, particularly for analysis of multiply glycosylated peptides (termed here middle-down glycoproteomics), has not been made. We therefore compared the performance of different ExD modes for middle-down glycopeptide analyses. We identified key features among the different dissociation modes and show that increased electron energy and supplemental activation provide the most useful data for middle-down glycopeptide analysis.

Graphical Abstract


Tandem MS Glycoproteomics Middle-down Electron-activated dissociation hotECD EThcD FTICR-MS 


Funding Information

This work was supported by NIH grants P41 GM104603 and S10 RR025082. Thermo Fisher Scientific provided access to the Orbitrap Fusion instrument.

Supplementary material

13361_2018_1909_MOESM1_ESM.pdf (2.3 mb)
ESM 1 (PDF 2402 kb)
13361_2018_1909_MOESM2_ESM.xlsx (953 kb)
ESM 2 (XLSX 952 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Department of Biochemistry, Center for Biomedical Mass SpectrometryBoston University School of MedicineBostonUSA
  2. 2.Department of ChemistryBoston UniversityBostonUSA
  3. 3.Program in BioinformaticsBoston UniversityBostonUSA
  4. 4.Boston University Medical CampusBostonUSA

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