Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.
IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.
Takahashi
Kazuo
K
Biomedical FT-ICR MS Laboratory, Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, Alabama 35294-0005, USA.
Wall
Stephanie B
SB
Suzuki
Hitoshi
H
Smith
Archer D
AD
Hall
Stacy
S
Poulsen
Knud
K
Kilian
Mogens
M
Mobley
James A
JA
Julian
Bruce A
BA
Mestecky
Jiri
J
Novak
Jan
J
Renfrow
Matthew B
MB
D000595:Amino Acid Sequence; D004583:Electrons; D005583:Fourier Analysis; D005922:Glomerulonephritis, IGA / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*
D006020:Glycopeptides; D007070:Immunoglobulin A; D011134:Polysaccharides
Glycobiology. 2008 Jun;18(6):456-62
18332077
J Am Soc Mass Spectrom. 2005 Jul;16(7):1060-6
15914017
Glycobiology. 2007 Apr;17(4):374-87
17215257
Mol Immunol. 2000 Dec;37(17):1047-56
11399322
Kidney Int. 2007 Jun;71(11):1148-54
17342176
Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6123-7
8016124
Biochem Biophys Res Commun. 2000 Apr 29;271(1):268-74
10777713
Kidney Int. 2001 Sep;60(3):969-73
11532091
Kidney Int. 2001 Mar;59(3):1077-85
11231363
J Clin Immunol. 2001 Sep;21(5):310-27
11720004
Anal Bioanal Chem. 2007 Nov;389(5):1397-407
17712550
Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):8894-9
19458039
Mol Cell Proteomics. 2006 May;5(5):923-34
16452088
J Am Soc Mass Spectrom. 2007 May;18(5):891-7
17350280
J Biol Chem. 2007 Apr 20;282(16):11893-903
17314104
J Mol Med (Berl). 1997 Aug;75(8):594-602
9297627
J Am Soc Mass Spectrom. 2009 Apr;20(4):639-51
19185509
J Clin Invest. 1999 Jul;104(1):73-81
10393701
Am J Med. 1988 Jan;84(1):129-32
3337116
Glycobiology. 2009 Apr;19(4):375-81
19095697
J Clin Invest. 2009 Jun;119(6):1668-77
19478457
Anal Chem. 2004 Jun 1;76(11):3112-8
15167790
Anal Chem. 2001 Sep 15;73(18):4530-6
11575803
Carbohydr Res. 2007 Nov 5;342(15):2322-5
17655836
Nat Chem Biol. 2007 Jun;3(6):339-48
17496889
Angew Chem Int Ed Engl. 2009;48(45):8526-8
19795429
Anal Chem. 2000 Oct 15;72(20):4778-84
11055690
Mol Cell Proteomics. 2007 Nov;6(11):1942-51
17673454
J Biol Chem. 2005 May 13;280(19):19136-45
15728186
J Clin Invest. 2008 Feb;118(2):629-39
18172551
AIDS. 2005 Mar 4;19(4):381-9
15750391
J Biol Chem. 1998 Jan 23;273(4):2260-72
9442070
Kidney Int. 1997 Aug;52(2):509-16
9264010
Anal Chem. 2007 Dec 15;79(24):9243-52
18020370
Anal Chem. 2009 Sep 15;81(18):7814-22
19689115
Contrib Nephrol. 1993;104:172-82
8325028
Adv Immunol. 2004;82:249-93
14975259
Mol Microbiol. 2002 Jan;43(1):147-57
11849543
J Proteome Res. 2010 Mar 5;9(3):1367-73
20104905
J Am Soc Nephrol. 1998 Apr;9(4):577-82
9555659
Anal Chem. 1999 Oct 15;71(20):4431-6
10546526
Anal Chem. 2009 Oct 1;81(19):7876-84
19722535
Anal Chem. 2010 Jan 15;82(2):579-84
20028093
Mol Cell Proteomics. 2009 Nov;8(11):2515-26
19674964
J Am Soc Mass Spectrom. 2009 Jun;20(6):1048-59
19278867
Contrib Nephrol. 2007;157:134-8
17495451
J Proteome Res. 2007 Nov;6(11):4230-44
17900180
Arthritis Rheum. 2007 Jan;56(1):21-9
17195187
Mol Immunol. 2007 Apr;44(10):2598-604
17275907
Science. 2001 Mar 23;291(5512):2370-6
11269318
Anal Chem. 2004 Apr 15;76(8):2231-8
15080732