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

Nature Methods volume 4, pages 437–444 (2007)Cite this article

Abstract

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.

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Figure 1: Detection of glycans on antibody arrays.
Figure 2: Optimization and characterization of chemical derivatization.
Figure 3: The effect of derivatization on antibody affinities.
Figure 4: Lectin binding profiles of native and enzyme-digested protein.
Figure 5: Profiling protein and glycan variation in cancer and control sera.

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Acknowledgements

We thank D. Mistry for assistance with the experiments and other members of the Laboratory of Cancer Immunodiagnostics at the Van Andel Research Institute for helpful interactions. We thank A. Rai (Memorial Sloan Kettering Cancer Center) and I. Goldstein (University of Michigan) for valuable input. We gratefully acknowledge the Van Andel Research Institute for support of this work.

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Author notes

  1. Tom LaRoche & Darren Hamelinck

    Present address: 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.).,

Authors and Affiliations

  1. Van Andel Research Institute, 333 Bostwick, Grand Rapids, 49503, Michigan, USA

    Songming Chen, Tom LaRoche, Darren Hamelinck, Derek Bergsma & Brian B Haab

  2. University of Michigan Medical Center, 1500 E. Hospital Drive, Ann Arbor, 48109, Michigan, USA

    Dean Brenner & Diane Simeone

  3. Evanston Northwestern Healthcare, 2100 Pfingstein Rd, Glenview, 60025, Illinois, USA

    Randall E Brand

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Correspondence to Brian B Haab.

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Competing interests

B.B.H. and S.C. are inventors on a patent application covering the technology described in this article. This technology has been licensed for commercialization by GenTel Biosciences (Madison, Wisconsin, USA).

Supplementary information

Supplementary Fig. 1

Chemical derivatization of antibodies to block lectin binding. (PDF 507 kb)

Supplementary Fig. 2

Optimization of antibody derivatization. (PDF 4163 kb)

Supplementary Fig. 3

Optimization of NaIO4 oxidation. (PDF 509 kb)

Supplementary Fig. 4

Effects of NaIO4 concentration on antibody retainment and background signals. (PDF 532 kb)

Supplementary Table 1

Lectins used in the experiments and their specificities. (PDF 133 kb)

Supplementary Table 2

Measured EC50s of 24 lectins for the glycans of spotted antibodies. (PDF 531 kb)

Supplementary Table 3

Antibodies and proteins used in the experiments. (PDF 21 kb)

Supplementary Methods (PDF 21 kb)

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Chen, S., LaRoche, T., Hamelinck, D. et al. Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays. Nat Methods 4, 437–444 (2007). https://doi.org/10.1038/nmeth1035

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