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Author Hinneburg, Hannes ♦ Stavenhagen, Kathrin ♦ Schweiger Hufnagel, Ulrike ♦ Pengelley, Stuart ♦ Jabs, Wolfgang ♦ Seeberger, Peter H. ♦ Silva, Daniel Varón ♦ Wuhrer, Manfred ♦ Kolarich, Daniel
Source SpringerLink
Content type Text
Publisher Springer US
File Format PDF
Copyright Year ©2015
Language English
Subject Domain (in DDC) Natural sciences & mathematics ♦ Chemistry & allied sciences
Subject Keyword Glycoproteomics ♦ Immunoglobulin ♦ Q-TOF ♦ Collision energy stepping CID ♦ Synthetic glycopeptides ♦ Glycopeptide ♦ N-glycan ♦ O-glycan ♦ Analytical Chemistry ♦ Biotechnology ♦ Organic Chemistry ♦ Proteomics ♦ Bioinformatics
Abstract In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control. Graphical Abstract ᅟ
ISSN 10440305
Age Range 18 to 22 years ♦ above 22 year
Educational Use Research
Education Level UG and PG
Learning Resource Type Article
Publisher Date 2016-01-04
Publisher Institution The American Society for Mass Spectrometry
Publisher Place New York
e-ISSN 18791123
Journal Journal of The American Society for Mass Spectrometry
Volume Number 27
Issue Number 3
Page Count 13
Starting Page 507
Ending Page 519


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Source: SpringerLink