title: Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: role of cysteine alkylation during sample processing.
abstract: Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236.
  
  
  CONCLUSIONS
  In this work, we studied the O-glycosylation in the hinge region of human immunoglobulin A1 (IgA1). Aberrant glycosylation of the protein plays a key role in the pathogenesis of IgA nephropathy. Thus identification of the O-glycan composition of IgA1 is important for a deeper understanding of the disease mechanism, biomarker discovery and validation, and implementation and monitoring of disease-specific therapies. We developed a new procedure for elucidating the heterogeneity of IgA1 O-glycosylation. After running a polyacrylamide gel electrophoresis under denaturing conditions, the heavy chain of IgA1 was subjected to in-gel digestion by trypsin. O-glycopeptides were separated from the digest on capillary columns using a microgradient chromatographic device (replacing commonly used liquid chromatographs) and subjected to MALDI-TOF/TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS) involving post-source decay fragmentation. We show that the complete modification of cysteines by iodoacetamide prior to electrophoresis is critical for successful MS/MS analyses on the way to deciphering the microheterogeneity of O-glycosylation in IgA1. Similarly, the removal of the excess of the reagent is equally important. The acquired MS/MS allowed assigning up to six O-glycosylation sites and identification of isomeric O-glycoforms. We show that our simplified approach is efficient and has a high potential to provide a method for the rapid assessment of IgA1 heterogeneity that is a less expensive and yet corroborating alternative to LC-(high-resolution)-MS protocols. The novelty and biological significance reside in the demonstration, for the first time, of the distribution of the most abundant isoforms of HR O-glycopeptides of IgA1. As another new feature, we introduce a software solution for the interpretation of MS/MS data of O-glycopeptide isoforms, which provides the possibility of fast and easier data processing. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.
authors.affiliation: Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic.
authors.forename: Vojtěch
authors.initials: V
authors.lastname: Franc
authors.forename: Pavel
authors.initials: P
authors.lastname: Řehulka
authors.forename: Martin
authors.initials: M
authors.lastname: Raus
authors.forename: Jiří
authors.initials: J
authors.lastname: Stulík
authors.forename: Jan
authors.initials: J
authors.lastname: Novak
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Renfrow
authors.forename: Marek
authors.initials: M
authors.lastname: Šebela
mesh_terms: D000478:Alkylation; D003545:Cysteine / Q000097:blood / Q000737:chemistry*; D005922:Glomerulonephritis, IGA / Q000097:blood; D006031:Glycosylation; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood / Q000737:chemistry*; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D007070:Immunoglobulin A; D003545:Cysteine
keywords: ACN; CAM; CHCA; DTT; ECD; ETD; FEP; FT-ICR; Fourier transform ion cyclotron resonance; Glycopeptide; HR; Human immunoglobulin A1 (IgA1); IAM; IgA; IgA nephropathy; IgAN; LC; MALDI-TOF; MS; MS/MS; Mass spectrometry; Microgradient separation; O-glycosylation; PAM; PSD; RPLC; TFA; acetonitrile; carbamidomethylation; dithiothreitol; electron capture dissociation; electron transfer dissociation; fluorinated ethylene propylene; hinge region; immunoglobulin A; iodoacetamide; liquid chromatography; mass spectrometry; matrix-assisted laser desorption/ionization time-of-flight; post-source decay; propionamidation; reversed-phase liquid chromatography; tandem mass spectrometry; trifluoroacetic acid; α-cyano-4-hydroxycinnamic acid
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