Abstract
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.
Highlights
Regulation of protein N-glycosylation is essential in human cells
We developed a protein aggregation capture (PAC)-based workflow for fast preparation of TMT-labeled N-glycopeptides (Fig. 1)
Using human lymphoma cell lysates (DG75 and Daudi), we evaluated and optimized cell lysis conditions, PAC bead types, ZIC-HILIC enrichment, and basic reverse phase (bRP) chromatography to achieve a shorter handling time and good recovery of N-glycopeptides (Supplementary Note 2)
Summary
Regulation of protein N-glycosylation is essential in human cells. 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. Significant improvements have been made with regard to methods for intact glycopeptide characterization, including glycopeptide enrichment[6,7,8], optimized MS acquisition strategies[8], advanced fragmentation techniques[9,10], and database search algorithms[11] These have helped address technical difficulties resulting from heterogeneous and complex glycopeptide structures and their less informative fragmentation behavior in tandem mass spectrometry (MS2). These developments have largely focused on increasing glycopeptide identification rates rather than on reliable, global glycopeptide quantification. Our findings uncover 2FF-sensitive N-glycosylation sites and reveal 2FF-mediated changes in N-glycosylation on key effectors in B-
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