Site-specific analysis of changes in the glycosylation of proteins in liver cirrhosis using data-independent workflow with soft fragmentation
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.
KeywordsData-independent analysis N-glycopeptide Fucosylation GP-SWATH
Compliance with ethical standards
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.