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.
Department of Oncology, Lombardi Comprehensive Cancer Center PSB GD9, Georgetown University, 3800 Reservoir Road NW, Washington, DC, 20057, USA.
Miloslav
M
Sanda
Department of Oncology, Lombardi Comprehensive Cancer Center PSB GD9, Georgetown University, 3800 Reservoir Road NW, Washington, DC, 20057, USA.
Lihua
L
Zhang
Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, 3800 Reservoir Road NW, Washington, DC, 20057, USA.
Nathan J
NJ
Edwards
Department of Oncology, Lombardi Comprehensive Cancer Center PSB GD9, Georgetown University, 3800 Reservoir Road NW, Washington, DC, 20057, USA. rg26@georgetown.edu.|Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, 3800 Reservoir Road NW, Washington, DC, 20057, USA. rg26@georgetown.edu.
Radoslav
R
Goldman
D001798:Blood Proteins / Q000737:chemistry; D006017:Glycolipids / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008099:Liver / Q000473:pathology / Q000503:physiopathology; D008103:Liver Cirrhosis / Q000503:physiopathology*
D001798:Blood Proteins; D006017:Glycolipids; C007790:fucosylglycolipids
Data-independent analysis; Fucosylation; GP-SWATH; N-glycopeptide