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

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 Burkitt's lymphoma cells, and determined complex site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…As such, stable isotope labeling strategies, including metabolic labeling and isobaric labeling, may prove particularly useful as quantitative glycoproteomics comes of age (63). Studies to investigate best practices for isobaric label-centric quantitative glycoproteomic data are emerging, including how to effectively choose ions for synchronous precursor selection for minimal co-isolation interference in multiplexed experiments (511)(512)(513)(514). The quantitative approaches selected for glycoproteomics experiments will continue to forge both data acquisition strategies to best acquire accurate quantitative data and also the informatic pipelines to extract and compare quantitative information.…”