Glyco-DIA

Glyco-DIA enables quantitative proteome-wide analysis of O-glycoproteins using a data-independent acquisition (DIA) LC–MS/MS workflow combined with in silico-boosted glycopeptide spectral libraries.

Key Features:

• In Silico-Boosted Glycopeptide Libraries: The spectral library includes sublibraries derived from human cell lines and human serum and encompasses 2,076 O-glycoproteins and 11,452 unique glycopeptide sequences.
• Coverage of Core1 O-Glycan Structures: The library covers the five most common core1 O-glycan structures.
• Data-Independent Acquisition LC–MS/MS: Uses a DIA strategy coupled with liquid chromatography and tandem mass spectrometry for direct analysis of O-glycoproteins on a proteome-wide scale.
• Single-Shot Analysis without Enrichment: Enables identification and quantification of glycopeptide sequences without prior enrichment, with a single-shot analysis identifying 269 glycopeptide sequences bearing up to five different core1 O-glycans from 159 glycoproteins in human serum.
• Quantification of Glycan Stoichiometries: Provides quantification of stoichiometries of glycan structures across the proteome while not providing direct unambiguous glycosylation site identification.
• Expandable DIA-Based Methodology: The DIA approach and spectral library are extendable to additional glycoproteomes beyond the initial human datasets.

Scientific Applications:

• Proteome-wide O-glycoproteomics: Enables direct, comprehensive quantitative analysis of O-glycoproteins across complex samples.
• Human Serum Studies: Supports large-scale analysis of human serum glycoproteomes.
• Biomarker Discovery: Facilitates discovery of glycosylation-based biomarkers.
• Disease Diagnostics: Supports applications in disease diagnostics that rely on O-glycoprotein quantification.
• Glycosylation Dynamics: Enables studies of glycosylation dynamics in health and disease through quantitative comparison of glycan stoichiometries.

Methodology:

Combines in silico-boosted glycopeptide spectral libraries (including sublibraries from human cell lines and human serum) with a data-independent acquisition (DIA) strategy coupled to liquid chromatography–tandem mass spectrometry to perform single-shot identification and quantification of glycopeptides and to quantify glycan stoichiometries.