Distinctive and Complementary MS2 Fragmentation Characteristics for Identification of Sulfated Sialylated N-Glycopeptides by nanoLC-MS/MS Workflow

  • Chu-Wei Kuo
  • Shih-Yun Guu
  • Kay-Hooi Khoo
Focus: Mass Spectrometry in Glycobiology and Related Fields: Research Article


High sensitivity identification of sulfated glycans carried on specific sites of glycoproteins is an important requisite for investigation of molecular recognition events involved in diverse biological processes. However, aiming for resolving site-specific glycosylation of sulfated glycopeptides by direct LC-MS2 sequencing is technically most challenging. Other than the usual limiting factors such as lower abundance and ionization efficiency compared to analysis of non-glycosylated peptides, confident identification of sulfated glycopeptides among the more abundant non-sulfated glycopeptides requires additional considerations in the selective enrichment and detection strategies. Metal oxide has been applied to enrich phosphopeptides and sialylated glycopeptides, but its use to capture sulfated glycopeptides has not been investigated. Likewise, various complementary MS2 fragmentation modes have yet to be tested against sialylated and non-sialylated sulfoglycopeptides due to limited appropriate sample availability. In this study, we have investigated the feasibility of sequencing tryptic sulfated N-glycopeptide and its MS2 fragmentation characteristics by first optimizing the enrichment methods to allow efficient LC-MS detection and MS2 analysis by a combination of CID, HCD, ETD, and EThcD on hybrid and tribrid Orbitrap instruments. Characteristic sulfated glyco-oxonium ions and direct loss of sulfite from precursors were detected as evidences of sulfate modification. It is anticipated that the technical advances demonstrated in this study would allow a feasible extension of our sulfoglycomic analysis to sulfoglycoproteomics.

Graphical Abstract


Sulfated sialylated glycopeptide Sulfoglycopeptides Sulfoglycomics Titanium dioxide EThcD HCD Orbitrap Fusion Tribrid Mass spectrometry 


Funding Information

This work was financially supported by an Academia Sinica Investigator Award grant to KKH and partly by the Research and Development Program of New Drugs and Vaccines for Critical Diseases of Academia Sinica and Ministry of Science and Technology (MOST 104-0210-01-09-02, MOST 105-0210-01-13-01, MOST 106-0210-01-15-02), Taiwan.

Supplementary material

13361_2018_1919_MOESM1_ESM.pdf (1.7 mb)
ESM 1 (PDF 1754 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Institute of Biological ChemistryAcademia SinicaTaipeiTaiwan

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