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bioRxiv · 10.1101/2020.05.20.105726

SugarQuant: a streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

Fang, P.·Ji, Y.·Silbern, I.·Doebele, C.·Ninov, M.·Lenz, C.·Oellerich, T.·Pan, K.-T.·Urlaub, H.
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Abstract

Site-specific regulation of protein N-glycosylation is essential in human cells. However, accurate quantification of glycosylation sites and their individual glycan moieties in a cell-wide manner is still technically challenging. Here, we introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising fast protein aggregation capture (PAC)-based sample preparation, optimized multi-notch MS3 LC-MS acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that allows for confident, global identification and quantification of intact glycopeptides in complex biological samples. PAC greatly reduces the overall samplehandling 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 significantly 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 intact glycopeptides by up to 22% when compared with one-step database search strategies. SugarQuant was applied to identify and quantify more than 5,000 unique glycoforms in Burkitts lymphoma cells, and determined complex site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

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Fang, P., Ji, Y., Silbern, I., Doebele, C., Ninov, M., Lenz, C., Oellerich, T., Pan, K.-T., Urlaub, H.. 2020-05-22. SugarQuant: a streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics. https://doi.org/10.1101/2020.05.20.105726

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