Clustered O-glycans of IgA1

Defining macro- and microheterogeneity by use of electron capture/transfer dissociation

Kazuo Takahashi, Stephanie B. Wall, Hitoshi Suzuki, Archer D. Smith IV, Stacy Hall, Knud Poulsen, Mogens Kilian, James A. Mobley, Bruce A. Julian, Jiri Mestecky, Jan Novak, Matthew B. Renfrow

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) Oglycans, 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 IgAspecific 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.

Original languageEnglish
Pages (from-to)2545-2557
Number of pages13
JournalMolecular and Cellular Proteomics
Volume9
Issue number11
DOIs
Publication statusPublished - 01-11-2010

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Immunoglobulin A
Polysaccharides
Macros
Electrons
Glycosylation
Hinges
Galactose
Ions
IgA-specific serine endopeptidase
Liquid chromatography
Liquid Chromatography
Peptide Hydrolases
Cyclotrons
Myeloma Proteins
Glycopeptides
Cyclotron resonance
Fourier Analysis
Threonine
Glomerulonephritis
Tandem Mass Spectrometry

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

Takahashi, Kazuo ; Wall, Stephanie B. ; Suzuki, Hitoshi ; Smith IV, Archer D. ; Hall, Stacy ; Poulsen, Knud ; Kilian, Mogens ; Mobley, James A. ; Julian, Bruce A. ; Mestecky, Jiri ; Novak, Jan ; Renfrow, Matthew B. / Clustered O-glycans of IgA1 : Defining macro- and microheterogeneity by use of electron capture/transfer dissociation. In: Molecular and Cellular Proteomics. 2010 ; Vol. 9, No. 11. pp. 2545-2557.
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abstract = "IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) Oglycans, 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 IgAspecific 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.",
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Takahashi, K, Wall, SB, Suzuki, H, Smith IV, AD, Hall, S, Poulsen, K, Kilian, M, Mobley, JA, Julian, BA, Mestecky, J, Novak, J & Renfrow, MB 2010, 'Clustered O-glycans of IgA1: Defining macro- and microheterogeneity by use of electron capture/transfer dissociation', Molecular and Cellular Proteomics, vol. 9, no. 11, pp. 2545-2557. https://doi.org/10.1074/mcp.M110.001834

Clustered O-glycans of IgA1 : Defining macro- and microheterogeneity by use of electron capture/transfer dissociation. / Takahashi, Kazuo; Wall, Stephanie B.; Suzuki, Hitoshi; Smith IV, Archer D.; Hall, Stacy; Poulsen, Knud; Kilian, Mogens; Mobley, James A.; Julian, Bruce A.; Mestecky, Jiri; Novak, Jan; Renfrow, Matthew B.

In: Molecular and Cellular Proteomics, Vol. 9, No. 11, 01.11.2010, p. 2545-2557.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Clustered O-glycans of IgA1

T2 - Defining macro- and microheterogeneity by use of electron capture/transfer dissociation

AU - Takahashi, Kazuo

AU - Wall, Stephanie B.

AU - Suzuki, Hitoshi

AU - Smith IV, Archer D.

AU - Hall, Stacy

AU - Poulsen, Knud

AU - Kilian, Mogens

AU - Mobley, James A.

AU - Julian, Bruce A.

AU - Mestecky, Jiri

AU - Novak, Jan

AU - Renfrow, Matthew B.

PY - 2010/11/1

Y1 - 2010/11/1

N2 - IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) Oglycans, 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 IgAspecific 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.

AB - IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) Oglycans, 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 IgAspecific 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.

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