Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays Songming Chen 1 , Tom LaRoche 1,4 , Darren Hamelinck 1,4 , Derek Bergsma 1 , Dean Brenner 2 , Diane Simeone 2 , Randall E Brand 3 & Brian B Haab 1 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. Glycan structures can be important determinants of many different biological processes, including protein-protein interactions, pro- tein trafficking and folding, immune recognition, cell adhesion and migration, and inter-cellular signaling. Alterations to glycan struc- tures can contribute to the development and progression of cancer and other diseases 1,2 . The ability to efficiently profile the variation in glycosylation in complex biological samples would be useful for a variety of purposes, such as to characterize disease-associated glycan alterations, to identify new diagnostic biomarkers or to study the factors that regulate glycan structures. Additionally, the targeting of glycan structures is an increasingly important ther- apeutic strategy 3 , and the efficient characterization of the breadth and diversity of proteins carrying certain glycans could aid those studies. Established methods of studying glycan structures, such as enzymatic removal of glycans followed by chromatographic separa- tion or mass spectrometry, give detailed information about glycans, but are not suitable for studies requiring reproducible measure- ments over many different samples or proteins. Affinity-based approaches, in which carbohydrate structures are detected by affinity reagents that bind to specific glycans, could be useful for such studies. Multiplexed methods that allow glycan measurements on many different, specific proteins could further add to the value of affinity-based glycan studies. Lectins—plant and animal proteins with natural carbohydrate binding functionality—have been valuable glycan affinity reagents in experimental formats such as affinity chromatography and electrophoresis 4 , detection of blots of separated glycoproteins 5 and in the capture or detection of proteins in microtiter plates to quantify glycans on specific proteins 6 . The use of multiple lectins in parallel can give a broad picture of the glycan structures present on proteins, as demonstrated in the use of microarrays of lectins to look at lectin-binding profiles of purified proteins 7–9 . Lectin-based glycan detection methods have been valuable for studying the roles of glycans in disease. Cancer-associated glycan variants have been found on major serum proteins such as a-fetoprotein 10 , hapto- globin 5,11 , a-1-acid glycoprotein 4 and a-1-antitrypsin 12 using lectin-affinity and immuno-affinity electrophoresis and blotting methods. Antibodies raised to particular glycan groups, such as the Thomsen-Friedenreich antigens 13 , the Lewis blood-group struc- tures 14 and underglycosylated MUC1 (ref. 15), also have been used to study the roles of glycans in cancer. We have developed a method that complements and adds to the existing glycan-detection technologies, based on the use of lectins to probe glycans on proteins captured by glycan-binding antibody arrays. This method allows the study of glycans on multiple, specific proteins captured directly from biological sam- ples. An essential preliminary step in the procedure is the chemical derivatization of the glycans on the spotted capture antibodies to prevent lectin binding to those glycans. We fully optimized and characterized that step for the detection of a wide variety of glycan structures. We demonstrate the use of multiple different lectins to obtain lectin-binding profiles of native transferrin captured under a variety of conditions, and we also demonstrate the characterization of the glycan variation on two serum proteins in pancreatic cancer and control patients. RECEIVED 5 DECEMBER 2006; ACCEPTED 12 MARCH 2007; PUBLISHED ONLINE 8 APRIL 2007; DOI:10.1038/NMETH1035 1 Van Andel Research Institute, 333 Bostwick, Grand Rapids, Michigan 49503, USA. 2 University of Michigan Medical Center, 1500 E. Hospital Drive, Ann Arbor, Michigan 48109, USA. 3 Evanston Northwestern Healthcare, 2100 Pfingstein Rd, Glenview, Illinois 60025, USA. 4 Present addresses: Wayne State University Medical School, 540 E. Canfield, Detroit, MI 48201, USA (T.L.), McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4L8 (D.H.). Correspondence should be addressed to B.B.H. (brian.haab@vai.org). NATURE METHODS | VOL.4 NO.5 | MAY 2007 | 437 ARTICLES