Abstract

Protein N-glycosylation patterns are known to show vast genetic as well as physiological and pathological variation and represent a large pool of potential biomarkers. Large-scale studies are needed for the identification and validation of biomarkers, and the analytical techniques required have recently been developed. Such methods have up to now mainly been applied to complex mixtures of glycoproteins in biofluids (e.g. plasma). Here, we analyzed N-glycosylation profiles of alpha1-antitrypsin (AAT) and immunoglobulin A (IgA) enriched fractions by 96-well microtitration plate based high-throughput immuno-affinity capturing and N-glycan analysis using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). Human plasma samples were from the Leiden Longevity Study comprising 2415 participants of different chronological and biological ages. Glycosylation patterns of AAT enriched fractions were found to be associated with chronological (calendar) age and they differed between females and males. Moreover, several glycans in the AAT enriched fraction were associated with physiological parameters marking cardiovascular and metabolic diseases. Pronounced differences were found between males and females in the glycosylation profiles of IgA enriched fractions. Our results demonstrate that large-scale immuno-affinity capturing of proteins from human plasma using a bead-based method combined with high-throughput N-glycan analysis is a powerful tool for the discovery of glycosylation-based biomarker candidates.

Highlights

  • Glycosylation occurs on more than 50% of human proteins [1] and proper glycosylation is essential for the survival of most multicellular organisms

  • Protein enrichment Glycoproteins were enriched from human plasma using highthroughput immuno-affinity capturing in a 96-well format

  • AntiAAT and anti-immunoglobulin A (IgA) antibody coated beads were applied to each of the wells of a filter plate, after which plasma was added to the wells

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Summary

Introduction

Glycosylation occurs on more than 50% of human proteins [1] and proper glycosylation is essential for the survival of most multicellular organisms. Protein Nglycosylation is very diverse, and more than 100 different Nglycans may be found on a specific protein [7]. The glycan signature, i.e. the total plasma N-glycome of an individual, reflects both genetic and physiological factors [8] and is highly reproducible in a given physiological state [9,10]. When the physiological state changes, e.g. due to aging or disease, the glycan pattern can change dramatically [9]. Due to the large variability of protein glycosylation and its reflection of physiological and pathological conditions, protein glycosylation patterns have been marked as a large field of potential biomarkers [11]

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