title: Generation of a natural glycan microarray using 9-fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag.
abstract: Glycan microarray technology has become a successful tool for studying protein-carbohydrate interactions, but a limitation has been the laborious synthesis of glycan structures by enzymatic and chemical methods. Here we describe a new method to generate quantifiable glycan libraries from natural sources by combining widely used protease digestion of glycoproteins and Fmoc chemistry. Glycoproteins including chicken ovalbumin, bovine fetuin, and horseradish peroxidase (HRP) were digested by Pronase, protected by FmocCl, and efficiently separated by 2D-HPLC. We show that glycans from HRP glycopeptides separated by HPLC and fluorescence monitoring retained their natural reducing end structures, mostly core alpha1,3-fucose and core alpha1,2-xylose. After simple Fmoc deprotection, the glycans were printed on NHS-activated glass slides. The glycans were interrogated using plant lectins and antibodies in sera from mice infected with Schistosoma mansoni, which revealed the presence of both IgM and IgG antibody responses to HRP glycopeptides. This simple approach to glycopeptide purification and conjugation allows for the development of natural glycopeptide microarrays without the need to remove and derivatize glycans and potentially compromise their reducing end determinants.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, O. Wayne Rollins Research Center, 1510 Clifton Road, Suite 4001, Atlanta, GA 30322, USA.
authors.forename: Xuezheng
authors.initials: X
authors.lastname: Song
authors.forename: Yi
authors.initials: Y
authors.lastname: Lasanajak
authors.forename: Carlos
authors.initials: C
authors.lastname: Rivera-Marrero
authors.forename: Anthony
authors.initials: A
authors.lastname: Luyai
authors.forename: Margaret
authors.initials: M
authors.lastname: Willard
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
mesh_terms: D000818:Animals; D005449:Fluorenes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006023:Glycoproteins / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D007202:Indicators and Reagents; D051379:Mice; D008810:Mice, Inbred C57BL; D046228:Microarray Analysis / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis*; D011402:Pronase / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005449:Fluorenes; D005456:Fluorescent Dyes; D006020:Glycopeptides; D006023:Glycoproteins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007202:Indicators and Reagents; D011134:Polysaccharides; C054007:1-(9-fluorenyl)methyl chloroformate; D006735:Horseradish Peroxidase; D011402:Pronase
references.citation: Methods Enzymol. 1994;247:87-106
references.pmid: 7898372
references.citation: Mol Cell Proteomics. 2007 Sep;6(9):1485-99
references.pmid: 17550893
references.citation: Nat Protoc. 2007;2(11):2747-58
references.pmid: 18007610
references.citation: J Am Chem Soc. 2004 Apr 21;126(15):4812-9
references.pmid: 15080685
references.citation: Curr Opin Struct Biol. 2003 Oct;13(5):637-45
references.pmid: 14568620
references.citation: Methods Enzymol. 1997;289:44-67
references.pmid: 9353717
references.citation: Chem Immunol Allergy. 2006;90:91-112
references.pmid: 16210905
references.citation: Chem Biol. 2004 Dec;11(12):1701-7
references.pmid: 15610854
references.citation: Carbohydr Res. 1986 Jun 1;149(1):65-83
references.pmid: 3731182
references.citation: Angew Chem Int Ed Engl. 2002 Sep 2;41(17):3180-2
references.pmid: 12207382
references.citation: Eur J Biochem. 1981 Apr;115(2):335-45
references.pmid: 7016535
references.citation: Glycobiology. 2006 Dec;16(12):21C-27C
references.pmid: 16971379
references.citation: J Am Chem Soc. 2004 Jul 21;126(28):8640-1
references.pmid: 15250702
references.citation: Carbohydr Res. 1981 Mar 2;89(2):221-8
references.pmid: 6113051
references.citation: Glycobiology. 2003 Mar;13(3):217-25
references.pmid: 12626421
references.citation: Methods Enzymol. 1982;83:235-41
references.pmid: 7098929
references.citation: Nat Methods. 2005 Nov;2(11):845-50
references.pmid: 16278655
references.citation: J Biol Chem. 1981 Jul 10;256(13):6633-40
references.pmid: 7240233
references.citation: Carbohydr Res. 1996 Jun 21;287(2):203-12
references.pmid: 8766207
references.citation: Biochem Soc Trans. 1988 Jun;16(3):264-5
references.pmid: 3181612
references.citation: Glycoconj J. 2008 Jan;25(1):15-25
references.pmid: 17763939
references.citation: Methods Enzymol. 2006;417:205-20
references.pmid: 17132507
references.citation: Methods Enzymol. 1997;289:221-45
references.pmid: 9353724
references.citation: Methods Mol Biol. 2008;494:187-208
references.pmid: 18726575
references.citation: Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17033-8
references.pmid: 15563589
references.citation: Adv Carbohydr Chem Biochem. 1994;50:277-310
references.pmid: 7942256
references.citation: Glycoconj J. 2001 Aug;18(8):573-87
references.pmid: 12376724
references.citation: Glycobiology. 1997 Sep;7(6):861-72
references.pmid: 9376689
references.citation: Cell Microbiol. 2004 Jan;6(1):13-22
references.pmid: 14678327
references.citation: Chem Soc Rev. 2008 Aug;37(8):1579-91
references.pmid: 18648683
references.citation: Glycobiology. 1997 Jul;7(5):663-77
references.pmid: 9254048
references.citation: Chem Biol. 2007 Jul;14(7):847-59
references.pmid: 17656321
references.citation: Chembiochem. 2004 Mar 5;5(3):379-82
references.pmid: 14997532
references.citation: Anal Biochem. 1994 Jan;216(1):165-70
references.pmid: 8135348
references.citation: Glycoconj J. 1995 Feb;12(1):84-93
references.pmid: 7540902
references.citation: Methods Enzymol. 2006;415:292-310
references.pmid: 17116481
references.citation: Exp Parasitol. 2000 Dec;96(4):202-12
references.pmid: 11162373
references.citation: Carbohydr Res. 2005 Sep 26;340(13):2111-22
references.pmid: 16026772
references.citation: Anal Biochem. 1995 Sep 20;230(2):229-38
references.pmid: 7503412
references.citation: J Proteome Res. 2009 Feb;8(2):425-6
references.pmid: 19133724
references.citation: Biochim Biophys Acta. 1997 Apr 17;1335(1-2):23-32
references.pmid: 9133639
references.citation: Anal Chem. 2007 May 15;79(10):3894-900
references.pmid: 17411071
references.citation: Int J Pept Protein Res. 1990 Mar;35(3):161-214
references.pmid: 2191922
references.citation: J Biol Chem. 1979 Oct 10;254(19):9795-9
references.pmid: 489569
references.citation: J Biol Chem. 1985 Apr 25;260(8):4688-93
references.pmid: 3988732
references.citation: Nat Rev Mol Cell Biol. 2004 Jul;5(7):582-8
references.pmid: 15232576
references.citation: Arch Biochem Biophys. 1995 Apr 20;318(2):465-75
references.pmid: 7733678
references.citation: Anal Biochem. 1998 Jan 15;255(2):183-7
references.pmid: 9451502
references.citation: Anal Chem. 2001 Dec 15;73(24):6063-9
references.pmid: 11791581
references.citation: Glycoconj J. 1998 Feb;15(2):203-6
references.pmid: 9557883
references.citation: Anal Chem. 2007 Nov 1;79(21):8107-13
references.pmid: 17922555
references.citation: FEBS Lett. 1976 Nov 15;72(1):117-20
references.pmid: 62678
references.citation: Nat Chem Biol. 2006 May;2(5):238-48
references.pmid: 16619023
references.citation: Biochem Soc Trans. 1984 Aug;12(4):607-10
references.pmid: 6208065
references.citation: Anal Chem. 2006 Sep 1;78(17):6081-7
references.pmid: 16944887
references.citation: Chem Biol. 2009 Jan 30;16(1):36-47
references.pmid: 19171304
references.citation: Genome Biol. 2002;3(12):REVIEWS1034
references.pmid: 12537579
references.citation: Glycobiology. 2001 Feb;11(2):149-63
references.pmid: 11287402
references.citation: J Biochem. 1975 Oct;78(4):687-96
references.pmid: 1213987