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.