Construction and Use of Glycan Microarrays

Christopher T. Campbell1, Yalong Zhang1, Jeffrey C. Gildersleeve1

1 National Cancer Institute, Frederick, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090228
Online Posting Date:  March, 2010
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Abstract

Glycosylation is an important post‐translational modification that influences many biological processes critical for development, normal physiologic function, and diseases. Unfortunately, progress toward understanding the roles of glycans in biology has been slow due to the challenges of studying glycans and the proteins that interact with them. Glycan microarrays provide a high‐throughput approach for the rapid analysis of carbohydrate‐macromolecule interactions. Protocols detailed here are intended to help laboratories with basic familiarity of DNA or protein microarrays to begin printing and performing assays using glycan microarrays. Basic and advanced data processing are also detailed, along with strategies for improving reproducibility of data collected with glycan arrays. Curr. Protoc. Chem Biol. 2:37‐53. © 2010 by John Wiley & Sons, Inc.

Keywords: glycosylation; microarray; neoglycoconjugate; carbohydrate‐dependent binding; serum antibody profile

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Glycan Microarrays
  • Basic Protocol 2: Glycan Array Profiling of Carbohydrate‐Binding Properties
  • Basic Protocol 3: Scanning and Data Analysis of Glycan Microarrays
  • Alternate Protocol 1: Advanced Processing and Methods: Extending the Dynamic Range of Pixel Intensity Measurements
  • Alternate Protocol 2: Normalize Slide Using Reference Sample
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of Glycan Microarrays

  Materials
  • Neoglycoproteins and/or glycoproteins (see protocol introduction above regarding sources)
  • Printing buffer (see recipe)
  • Controls: unmodified BSA and HSA (negative controls) and BSA conjugated with Cy3 or Alexa Fluor 647
  • 384‐well V‐bottom sample plates with lids (X6004, Genetix)
  • Aluminum plate seals (07‐200‐683, Costar)
  • Centrifuge with microtiter plate carrier
  • Robotic Microarray Printer (MicroGrid II, Genomic Solutions; http://bioinformatics.genomicsolutions.com/)
  • Microscope (dissecting or basic optical transmission microscope)
  • Pins (Stealth Microspotting SMP3 Pins, Arrayit; http://www.arrayit.com/)
  • Epoxide Coated Slides (SuperEpoxy 2 Premium Microarray Substrates, Arrayit; http://www.arrayit.com/)
  • Hygrometer

Basic Protocol 2: Glycan Array Profiling of Carbohydrate‐Binding Properties

  Materials
  • Glycan arrays ( protocol 1)
  • Blocking buffer: 3% (w/v) bovine serum albumin (BSA, Ig‐free; Sigma, cat. no. A‐3059) in 1× phosphate‐buffered saline (PBS; see recipe for 10×)
  • PBST array wash buffer (see recipe)
  • Appropriate incubation buffer for sample, consisting of BSA and PBST (Table 9.2.2800)
  • Sample (see Table 9.2.2800 for appropriate concentrations)
  • Reference sample, e.g., pooled serum or biotinylated lectins known to bind glycans on the array
  • Secondary antibody and/or Cy3‐conjugated streptavidin (Jackson ImmunoResearch) and appropriate buffer (Table 9.2.2800)
  • Microscope (dissecting or basic optical transmission microscope)
  • Slide module (ProPlate, Invitrogen)
  • Adhesive seals for slide module (ProPlate, Invitrogen)
  • Orbital shaker
  • 50‐ml conical centrifuge tubes
  • Centrifuge
    Table 9.2.2   MaterialsRecommended Buffers and Starting Dilutions of Samples for Incubation on Glycan MicroarrayRecommended Secondary Reagents and Conditions

    Sample type Incubation buffer a Concentration Conditions
    Lectin 1% BSA in PBST 1‐50 µg/ml Room temperature for 2 hr
    Monoclonal antibody 3% BSA in PBST 1‐50 µg/ml 37°C while gently shaken for 2‐4 hr
    Serum antibodies 3% BSA in PBST 1:50 to 1:200 37°C while gently shaken for 4 hr
    Sample type Secondary label Buffer Concentration Conditions
    Biotinylated lectin Cy3‐Streptavidin 1% BSA in PBS 2 µg/ml Room temperature for 2 hr
    Monoclonal antibody Monoclonal antibody specific for species and isotype of primary Ab 3% BSA in PBS 2 µg/ml 37°C while gently shaken for 2 hr
    Human serum antibodies Monoclonal anti‐human IgG, IgM, and/or IgA 3% HSA + 1% BSA in PBS 2 µg/ml 37 °C while gently shaken for 2 hr

     aPBST: PBST: see recipe for PBST array wash buffer in Reagents and Solutions.
    Table 9.2.3   MaterialsRecommended Buffers and Starting Dilutions of Samples for Incubation on Glycan MicroarrayRecommended Secondary Reagents and Conditions

    Sample type Incubation buffer a Concentration Conditions
    Lectin 1% BSA in PBST 1‐50 µg/ml Room temperature for 2 hr
    Monoclonal antibody 3% BSA in PBST 1‐50 µg/ml 37°C while gently shaken for 2‐4 hr
    Serum antibodies 3% BSA in PBST 1:50 to 1:200 37°C while gently shaken for 4 hr
    Sample type Secondary label Buffer Concentration Conditions
    Biotinylated lectin Cy3‐Streptavidin 1% BSA in PBS 2 µg/ml Room temperature for 2 hr
    Monoclonal antibody Monoclonal antibody specific for species and isotype of primary Ab 3% BSA in PBS 2 µg/ml 37°C while gently shaken for 2 hr
    Human serum antibodies Monoclonal anti‐human IgG, IgM, and/or IgA 3% HSA + 1% BSA in PBS 2 µg/ml 37 °C while gently shaken for 2 hr

NOTE: Prepare all solutions fresh on the day of the experiment.NOTE: All BSA and HSA must be globulin‐free to avoid high levels of background binding. Albumin that has not been sufficiently purified contains globulins, which will interfere in the assay. Other sources of albumin purified by gel electrophoresis may be substituted after confirming that they do not contain contaminating immunoglobulins that bind to the array.

Basic Protocol 3: Scanning and Data Analysis of Glycan Microarrays

  Materials
  • Arrays ( protocol 2)
  • Fluorescence scanner (GenePix 4000A, Molecular Devices)
  • Image processing software (GenePix Pro 6.0, Molecular Devices)
  • Microsoft Excel
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Figures

Videos

Literature Cited

Literature Cited
   Ban, L. and Mrksich, M. 2008. On‐chip synthesis and label‐free assays of oligosaccharide arrays. Angew. Chem. Int. Ed. Engl. 47:3396‐3399.
   Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan, M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Skehel, J.J., van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C.H., and Paulson, J.C. 2004. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U.S.A. 101:17033‐17038.
   Blixt, O., Han, S., Liao, L., Zeng, Y., Hoffmann, J., Futakawa, S., and Paulson, J.C. 2008a. Sialoside analogue arrays for rapid identification of high affinity siglec ligands. J. Am. Chem. Soc. 130:6680‐6681.
   Blixt, O., Hoffmann, J., Svenson, S., and Norberg, T. 2008b. Pathogen specific carbohydrate antigen microarrays: A chip for detection of Salmonella O‐antigen specific antibodies. Glycoconj. J. 25:27‐36.
   Culf, A.S., Cuperlovic‐Culf, M., and Ouellette, R.J. 2006. Carbohydrate microarrays: Survey of fabrication techniques. OMICS 10:289‐310.
   Cummings, R.D. 2009. The repertoire of glycan determinants in the human glycome. Mol. Biosyst. 5:1087‐1104.
   Deng, Y., Zhu, X.Y., Kienlen, T., and Guo, A. 2006. Transport at the air/water interface is the reason for rings in protein microarrays. J. Am. Chem. Soc. 128:2768‐69.
   Freedman, M.S., Laks, J., Dotan, N., Altstock, R.T., Dukler, A., and Sindic, C.J. 2009. Anti‐alpha‐glucose‐based glycan IgM antibodies predict relapse activity in multiple sclerosis after the first neurological event. Mult. Scler. 15:422‐430.
   Gildersleeve, J.C., Oyelaran, O., Simpson, J.T., and Allred, B. 2008. Improved procedure for direct coupling of carbohydrates to proteins via reductive amination. Bioconjug. Chem. 19:1485‐1490.
   Gu, Q., Sivanandam, T.M., and Kim, C.A. 2006. Signal stability of Cy3 and Cy5 on antibody microarrays. Proteome Sci. 4:21.
   Kamena, F., Tamborrini, M., Liu, X., Kwon, Y.U., Thompson, F., Pluschke, G., and Seeberger, P.H. 2008. Synthetic GPI array to study antitoxic malaria response. Nat. Chem. Biol. 4:238‐240.
   Liang, P.H., Wu, C.Y., Greenberg, W.A., and Wong, C.H. 2008. Glycan arrays: Biological and medical applications. Curr. Opin. Chem. Biol. 12:86‐92.
   Liu, Y., Feizi, T., Campanero‐Rhodes, M.A., Childs, R.A., Zhang, Y., Mulloy, B., Evans, P.G., Osborn, H.M., Otto, D., Crocker, P.R., and Chai, W. 2007. Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide‐protein interactions. Chem. Biol. 14:847‐859.
   Liu, Y., Palma, A.S. and Feizi, T. 2009. Carbohydrate microarrays: Key developments in glycobiology. Biol. Chem. 390:647‐656.
   Lyng, H., Badiee, A., Svendsrud, D.H., Hovig, E., Myklebost, O., and Stokke, T. 2004. Profound influence of microarray scanner characteristics on gene expression ratios: Analysis and procedure for correction. BMC Genomics 5:10.
   Manimala, J.C., Roach, T.A., Li, Z., and Gildersleeve, J.C. 2006. High‐throughput carbohydrate microarray analysis of 24 lectins. Angew. Chem. Int. Ed. Engl. 45:3607‐3610.
   Manimala, J.C., Roach, T.A., Li, Z., and Gildersleeve, J.C. 2007. High‐throughput carbohydrate microarray profiling of 27 antibodies demonstrates widespread specificity problems. Glycobiology 17:17C‐23C.
   Oyelaran, O. and Gildersleeve, J.C. 2009. Glycan arrays: Recent advances and future challenges. Curr. Opin. Chem. Biol. 13:406‐413.
   Oyelaran, O., Li, Q., Farnsworth, D., and Gildersleeve, J.C. 2009. Microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies. J. Proteome Res. 8:3529‐3538.
   Park, S. and Shin, I. 2007. Carbohydrate microarrays for assaying galactosyltransferase activity. Org. Lett. 9:1675‐678.
   Parthasarathy, N., Saksena, R., Kovac, P., Deshazer, D., Peacock, S.J., Wuthiekanun, V., Heine, H.S., Friedlander, A.M., Cote, C.K., Welkos, S.L., Adamovicz, J.J., Bavari, S., and Waag, D.M. 2008. Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies. Carbohydr. Res. 2008 Jun 14. [Epub ahead of print]
   Pohl, N.L. 2008. Fluorous tags catching on microarrays. Angew. Chem. Int. Ed. Engl. 47:3868‐3870.
   Ratner, D.M., Adams, E.W., Su, J., O'Keefe, B.R., Mrksich, M., and Seeberger, P.H. 2004. Probing protein‐carbohydrate interactions with microarrays of synthetic oligosaccharides. Chembiochem 5:379‐382.
   Roy, R., Katzenellenbogen, E., and Jennings, H.J. 1984. Improved procedures for the conjugation of oligosaccharides to protein by reductive amination. Can. J. Biochem. Cell Biol. 62:270‐275.
   Schallus, T., Jaeckh, C., Feher, K., Palma, A.S., Liu, Y., Simpson, J.C., Mackeen, M., Stier, G., Gibson, T.J., Feizi, T., Pieler, T., and Muhle‐Goll, C. 2008. Malectin: A novel carbohydrate‐binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N‐glycosylation. Mol. Biol. Cell 19:3404‐3414.
   Seeberger, P.H. 2008. Automated oligosaccharide synthesis. Chem. Soc. Rev. 37:19‐28.
   Seow, C.H., Stempak, J.M., Xu, W., Lan, H., Griffiths, A.M., Greenberg, G.R., Steinhart, A.H., Dotan, N., and Silverberg, M.S. 2009. Novel anti‐glycan antibodies related to inflammatory bowel disease diagnosis and phenotype. Am. J. Gastroenterol. 104:1426‐1434.
   Song, E.H. and Pohl, N.L. 2009. Carbohydrate arrays: Recent developments in fabrication and detection methods with applications. Curr. Opin. Chem. Biol. 13:626‐632.
   Song, X., Xia, B., Stowell, S.R., Lasanajak, Y., Smith, D.F., and Cummings, R.D. 2009. Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem. Biol. 16:36‐47.
   Wang, C.C., Huang, Y.L., Ren, C.T., Lin, C.W., Hung, J.T., Yu, J.C., Yu, A.L., Wu, C.Y., and Wong, C.H. 2008. Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer. Proc. Natl. Acad. Sci. U.S.A. 105:11661‐11666.
   Wang, D., Liu, S., Trummer, B.J., Deng, C., and Wang, A. 2002. Carbohydrate microarrays for the recognition of cross‐reactive molecular markers of microbes and host cells. Nat. Biotechnol. 20:275‐281.
   Wong, S.Y. 1995. Neoglycoconjugates and their applications in glycobiology. Curr. Opin. Struct. Biol. 5:599‐604.
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