Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays

• Published in: Nature methods Vol. 4; no. 5; pp. 437 - 444
• Main Authors: Haab, Brian B, Chen, Songming, LaRoche, Tom, Hamelinck, Darren, Bergsma, Derek, Brenner, Dean, Simeone, Diane, Brand, Randall E
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.05.2007
• Subjects: Analysis; Antibodies - chemistry; Antigens, Neoplasm - chemistry; Carbohydrates; Carcinoembryonic Antigen - chemistry; Humans; Lectins - chemistry; Methods; Microarray Analysis - methods; Mucin-1; Mucins - chemistry; Pancreatic cancer; Pancreatic Neoplasms - blood; Pancreatic Neoplasms - metabolism; Polysaccharides; Polysaccharides - chemistry; Polysaccharides - immunology; Probes; Protein binding; Protein microarrays; Protein Processing, Post-Translational; Proteins; Transferrin - chemistry; United States
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth1035

Carbohydrate post-translational modifications on proteins are important determinants of protein function in both normal and disease biology. We have developed a method to allow the efficient, multiplexed study of glycans on individual proteins from complex mixtures, using antibody microarray capture of multiple proteins followed by detection with lectins or glycan-binding antibodies. Chemical derivatization of the glycans on the spotted antibodies prevented lectin binding to those glycans. Multiple lectins could be used as detection probes, each targeting different glycan groups, to build up lectin binding profiles of captured proteins. By profiling both protein and glycan variation in multiple samples using parallel sandwich and glycan-detection assays, we found cancer-associated glycan alteration on the proteins MUC1 and CEA in the serum of pancreatic cancer patients. Antibody arrays for glycan detection are highly effective for profiling variation in specific glycans on multiple proteins and should be useful in diverse areas of glycobiology research.