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Analytical and Bioanalytical Chemistry

, Volume 409, Issue 2, pp 619–627 | Cite as

Site-specific analysis of changes in the glycosylation of proteins in liver cirrhosis using data-independent workflow with soft fragmentation

  • Miloslav Sanda
  • Lihua Zhang
  • Nathan J. Edwards
  • Radoslav Goldman
Research Paper
Part of the following topical collections:
  1. Glycomics, Glycoproteomics and Allied Topics

Abstract

Cirrhosis of the liver is associated with increased fucosylation of proteins in the plasma. We describe a data-independent (DIA) strategy for comparative analysis of the site-specific glycoforms of plasma glycoproteins. A library of 161 glycoforms of 25 N-glycopeptides was established by data-dependent LC-MS/MS analysis of a tryptic digest of 14 human protein groups retained on a multiple affinity removal column. The collision-induced dissociation conditions were adjusted to maximize the yield of selective Y-ions which were quantified by a data-independent mass spectrometry workflow using a 10-Da acquisition window. Using this workflow, we quantified 125 glycoforms of 25 glycopeptides, covering 10 of the 14 proteins, without any further glycopeptide enrichment. Comparison of the proteins in the plasma of healthy controls and cirrhotic patients shows an average 1.5-fold increase in the fucosylation of bi-antennary glycoforms and 3-fold increase in the fucosylation of tri- and tetra- antennary glycoforms. These results show that the adjusted glycopeptide DIA workflow using soft collision-induced fragmentation of glycopeptides is suitable for site-specific analysis of protein glycosylation in complex mixtures of analytes without glycopeptide enrichment.

Keywords

Data-independent analysis N-glycopeptide Fucosylation GP-SWATH 

Notes

Compliance with ethical standards

Funding

This work was supported by National Institutes of Health Grants UO1 CA168926, UO1 CA171146, and RO1 CA135069 (to R.G.) and CCSG Grant P30 CA51008 (to Lombardi Comprehensive Cancer Center supporting the Proteomics and Metabolomics Shared Resource).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval and consent to participate

All the participants were enrolled and signed informed consent under protocols approved by the MedStar Health Research Institute—Georgetown University Oncology Institutional Review Board (IRB) under IRB # 2014-0804. All bio-specimens are de-identified and may not be linked to identifiable individual private information.

Supplementary material

216_2016_41_MOESM1_ESM.pdf (153 kb)
ESM 1 (PDF 153 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Miloslav Sanda
    • 1
  • Lihua Zhang
    • 1
  • Nathan J. Edwards
    • 2
  • Radoslav Goldman
    • 1
    • 2
  1. 1.Department of Oncology, Lombardi Comprehensive Cancer Center PSB GD9Georgetown UniversityWashingtonUSA
  2. 2.Department of Biochemistry and Molecular & Cellular BiologyGeorgetown UniversityWashingtonUSA

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