Analysis of heterogeneous hinge-region O-glycosylation of human IgA1 using MALDI-TOF/TOF mass spectrometry

Vojtěch Franc¹, Pavel Řehulka^2*, Martin Raus³, Jiří Stulík², Jan Novak^4**, Matthew B. Renfrow⁵, and Marek Šebela^1,3***

¹Department 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;
²Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, Třebešská 1575, CZ-500 01 Hradec Králové, Czech Republic;
³Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11,
CZ-783 71 Olomouc, Czech Republic;

⁴Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;

⁵Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA

Changes in the glycosylation patterns of various glycoproteins are associated with several diseases. Hence determining disease-associated glycosylation patterns and heterogeneity provides a better understanding of disease mechanisms. This work focuses on the O-glycosylation of immunoglobulin A1 (IgA1), where aberrant glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). IgA1 hinge region carries 3-6 O-glycans consisting of N-acetylgalactosamine (GalNAc) with galactose (Gal); both glycans 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 hinge-region glycopeptides by MALDI-TOF/TOF mass spectrometry (MS) as a novel strategy. IgA1 hinge-region 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, which allowed assigning up to six O-glycosylation sites and suggested possible isomeric O-glycoforms. 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.