A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

2020 | journal article; research paper. A publication with affiliation to the University of Göttingen.

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​A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics​
Fang, P.; Ji, Y.; Silbern, I.; Doebele, C. ; Ninov, M.; Lenz, C.   & Oellerich, T. et al.​ (2020) 
Nature Communications11(1).​ DOI: https://doi.org/10.1038/s41467-020-19052-w 

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Fang, Pan; Ji, Yanlong; Silbern, Ivan; Doebele, Carmen ; Ninov, Momchil; Lenz, Christof ; Oellerich, Thomas; Pan, Kuan-Ting; Urlaub, Henning 
Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.
Issue Date
Nature Communications 
SFB 1286: Quantitative Synaptologie 
SFB 1286 | A06: Mitochondrienfunktion und -umsatz in Synapsen 
Working Group
RG Urlaub (Bioanalytische Massenspektrometrie) 
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