Development and application of GlycanDIA workflow for glycomic analysis

Area of Science:

• Glycomics and Mass Spectrometry
• Cellular Biology and Disease Mechanisms

Background:

• Glycans form the cell surface glycocalyx, regulating cellular processes.
• Altered glycosylation patterns are implicated in disease initiation and progression.
• Existing glycomic methods face limitations in sensitivity and precision for complex samples.

Purpose of the Study:

• To introduce GlycanDIA, a novel data-independent acquisition (DIA)-based glycomic workflow.
• To develop GlycanDIA Finder, a generic search engine for confident glycan identification from DIA data.
• To profile and compare N-glycans on RNA and proteins, including low-abundance species.

Main Methods:

• Implementation of a DIA-based glycomic workflow combining higher energy collisional dissociation (HCD)-MS/MS and staggered windows.
• Development of the GlycanDIA Finder search engine with iterative decoy searching for enhanced glycan identification.
• Application of the workflow to distinguish glycan composition and isomers (N-glycans, O-glycans, HMOs) and profile glycoRNA.

Main Results:

• GlycanDIA demonstrates high sensitivity and precision in glycan identification and quantification.
• The workflow successfully differentiates glycan isomers and identifies low-abundant modified glycans.
• Analysis of glycoRNA revealed distinct N-glycan abundance compared to protein-glycans, with tissue-specific variations.

Conclusions:

• GlycanDIA offers a significant advancement for sensitive and precise glycomic analysis.
• The developed workflow enables comprehensive profiling of N-glycans on RNA molecules.
• Distinct glycoRNA profiles suggest unique functional roles for RNA-glycans in biological processes and tissues.