Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum.
The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments, preventing peptide identification. In addition, O-linked structures also undergo a gas-phase rearrangement reaction, which eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we report the enrichment and mass spectrometric analysis of proteins from bovine serum bearing Galbeta1-3GalNAcalpha (mucin core-1 type) structures and the analysis of O-linked glycopeptides utilizing electron transfer dissociation and high resolution, high mass accuracy precursor ion measurements. Electron transfer dissociation (ETD) analysis of intact glycopeptides provided sufficient information for the identification of several glycosylation sites. However, glycopeptides frequently feature precursor ions of low charge density (m/z > approximately 850) that will not undergo efficient ETD fragmentation. Exoglycosidase digestion was utilized to reduce the mass of the molecules while retaining their charge. ETD analysis of species modified by a single GalNAc at each site was significantly more successful in the characterization of multiply modified molecules. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the enrichment method as well as the ETD analysis.
Darula
Zsuzsanna
Z
Proteomics Research Group, Biological Research Center of the Hungarian Academy of Sciences, Szeged P. O. Box 521, Szeged H-6701, Hungary.
Medzihradszky
Katalin F
KF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000509:alpha-Fetoproteins / Q000737:chemistry
D006020:Glycopeptides; D009077:Mucins; D010455:Peptides; D000509:alpha-Fetoproteins
Anal Chem. 2000 Feb 1;72(3):563-73
10695143
Nat Methods. 2008 May;5(5):405-7
18425140
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Aug 7;840(1):29-36
16843073
J Biol Chem. 1954 Jun;208(2):669-78
13174577
Mol Cell Proteomics. 2007 Nov;6(11):1942-51
17673454
J Biol Chem. 1999 Jun 25;274(26):18165-72
10373415
J Chromatogr A. 2006 Nov 3;1132(1-2):165-73
16919642
Annu Rev Biochem. 1997;66:315-35
9242909
Eur J Biochem. 1993 Nov 15;218(1):1-27
8243456
Proc Natl Acad Sci U S A. 1977 Jan;74(1):134-8
264667
Nat Biotechnol. 2003 Jun;21(6):660-6
12754519
Mol Cell Proteomics. 2005 Feb;4(2):107-19
15561728
Mol Cell Proteomics. 2006 May;5(5):923-34
16452088
Glycobiology. 2006 Jan;16(1):46-53
16177266
Mol Cell Proteomics. 2006 Oct;5(10):1957-67
16760258
Mol Cell Proteomics. 2007 Oct;6(10):1778-87
17623646
Eur J Mass Spectrom (Chichester). 2005;11(5):497-511
16322656
J Biol Chem. 1987 Feb 25;262(6):2768-79
3546295
Anal Chem. 1997 Oct 1;69(19):3986-94
9322435
Biochemistry. 1994 Nov 22;33(46):13524-30
7947762
J Lipid Res. 1987 Dec;28(12):1405-9
3123586
J Biol Chem. 2000 Sep 15;275(37):28569-74
10878002
Anal Biochem. 1990 Dec;191(2):262-7
2085172
J Am Soc Mass Spectrom. 1996 Apr;7(4):319-28
24203358
J Biol Chem. 2007 Jul 6;282(27):19463-72
17500062
J Biol Chem. 1995 Feb 10;270(6):2541-9
7852316
J Proteome Res. 2006 Mar;5(3):701-8
16512686
J Hepatol. 2009 Mar;50(3):592-603
19157620
Biomed Environ Mass Spectrom. 1990 Dec 5;19(12):777-81
1708302
Annu Rev Biochem. 2003;72:643-91
12676797
Anal Chem. 2003 May 15;75(10):2355-61
12918977
Biochem J. 1991 Apr 15;275 ( Pt 2):529-34
2025231
J Proteomics. 2008 Aug 21;71(3):304-17
18638581
Methods Enzymol. 2005;405:139-71
16413314
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Sep 5;776(2):149-60
12137996
J Biol Chem. 1992 Apr 25;267(12):8153-60
1569071
Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9528-33
15210983
J Proteome Res. 2008 Aug;7(8):3127-36
18613715
Glycobiology. 2005 Feb;15(2):153-64
15385431
J Proteome Res. 2004 May-Jun;3(3):556-66
15253437
J Proteome Res. 2009 Feb;8(2):493-501
19067536
Biochemistry. 1997 Aug 19;36(33):10200-12
9254618
Glycobiology. 1993 Apr;3(2):97-130
8490246
Mol Cell Proteomics. 2007 Apr;6(4):708-16
17220477
Biochemistry. 1998 Jan 6;37(1):408-16
9425062
J Proteome Res. 2009 Feb;8(2):631-42
19093876
Biochim Biophys Acta. 1999 Dec 6;1473(1):4-8
10580125
Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):8894-9
19458039
Mol Cell Proteomics. 2009 Apr;8(4):791-8
19088065
BMC Bioinformatics. 2008 Feb 18;9:101
18282281
Eur J Mass Spectrom (Chichester). 2004;10(5):625-38
15531796