Proteomics

Dataset Information

2

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 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 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo sapiens  

TISSUE(S): Tissue Not Applicable To Dataset

DISEASE(S): Not Available

SUBMITTER: Pan Fang  

LAB HEAD: Kuan-Ting Pan

PROVIDER: PXD018349 | Pride | 2020-08-24

REPOSITORIES: Pride

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Publications

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

Fang Pan P   Ji Yanlong Y   Silbern Ivan I   Doebele Carmen C   Ninov Momchil M   Lenz Christof C   Oellerich Thomas T   Pan Kuan-Ting KT   Urlaub Henning H  

Nature communications 20201019 1


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  ...[more]

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