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Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography
Published on: January 18, 2020
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Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications
Ana Momčilović1, Noortje de Haan1, Agnes L Hipgrave Ederveen1
1Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
Analytical Chemistry
|February 25, 2020
Summary
This study introduces a novel, high-throughput method for analyzing immunoglobulin (Ig) glycopeptides in serum. The technique enables simultaneous quantification of N- and O-glycopeptides from IgG and IgA, revealing new age-related glycosylation patterns.
Area of Science:
- Biochemistry
- Immunology
- Analytical Chemistry
Background:
- Immunoglobulin (Ig) glycosylation significantly impacts Ig turnover and effector functions.
- Large-scale Ig glycosylation profiling is complex due to isotype/subclass diversity, concentration variations, and multiple glycosylation sites.
- Existing methods face challenges in high-throughput analysis of diverse Ig glycopeptides.
Purpose of the Study:
- To develop and validate a high-throughput nanoLC-MS method for simultaneous analysis of IgG and IgA glycopeptides.
- To apply this method to a large cohort of healthy donors for comprehensive glycopeptide profiling.
- To identify novel associations between Ig glycosylation and demographic factors like age.
Main Methods:
- Simultaneous enrichment of IgG and IgA from small serum volumes using a 96-well plate format.
- One-step sample preparation including denaturation, reduction, and alkylation, followed by trypsin digestion.
- High-throughput nano-liquid chromatography (LC)-mass spectrometry (MS) with semiautomated data analysis.
Main Results:
- Reliable quantification of 143 unique N- and O-glycopeptides from IgG1, IgG2/3, IgG4, IgA1, IgA2, and dimeric IgA.
- Demonstrated high method repeatability using pooled plasma and serum standards.
- Identified novel associations between IgA glycosylation and age, and confirmed known IgG Fc glycosylation-age associations.
Conclusions:
- The developed nanoLC-MS method offers a simplified, high-throughput approach for multiprotein plasma glycoproteomics.
- This method facilitates large-scale glycopeptide analysis, advancing our understanding of Ig glycosylation in health and disease.
- The findings highlight the potential of glycoproteomics in discovering new biomarkers and understanding biological processes.
