Boronic Acid Lectin Affinity Chromatography (BLAC). 2. Affinity Micropartitioning-Mediated Comparative Glycosylation Profiling

As a continuation of our work on boronic acid lectin affinity chromatography (BLAC), in this paper we introduce an automated affinity micropartitioning approach using combined boronic acid and concanavalin A (BLAC/Con A) resin-filled micropipette tips to isolate and enrich human serum glycoproteins. The N-linked oligosaccharides of the partitioned glycoproteins were removed by PNGase F enzyme digestion, followed by 8-aminopyrene-1,3,6-trisulfonic acid labeling. Capillary gel electrophoresis with blue LED-induced fluorescence detection was applied in a multiplexed format for comparative glycan profiling. The efficiency of BLAC affinity micropartitioning was compared with that of the individual lectin and pseudolectin affinity enrichment. Finally, we report on our findings in glycosylation differences in human serum samples from healthy and prostate cancer patients by applying BLAC/Con A micropipette tip-based enrichment and comparative multicapillary gel electrophoresis analysis of the released and labeled glycans.