Podium Presentation in Room 2 on Thursday at 11:20 (Chair: Richard Drake)
Authors: Hsiao-Fan Chen (1), San-Yuan Wang (2), Chih-Chin Kao(3,*), Isabel I-Lin Tsai (1,2,*)
INTRODUCTION: Immunoglobulin A nephropathy (IgAN) is an autoimmune disease which is characterized by the deposition of IgA and IgA-IgG/IgA-IgA immune complex in the glomerular mesangial cells. It was found that galactose deficiency (Gd) of O-linked glycan at the human IgA subclass, IgA1, hinge region (HR) makes IgA1 as an autoantigen which induced the generation of autoantibodies. Mass spectrometry-based methods, such as MALDI-TOFMS or nanoLC-high resolution mass spectrometry have been used for discovering the abnormality of IgA1 HR O-glycan.
OBJECTIVES: In this study, we aimed to develop a simple method that used UHPLC coupled with triple quadrupole mass spectrometry to monitor Gd-IgA1 from human bio-fluids.
METHODS: We used peptide M affinity beads to purify human IgA from plasma followed by a simple tryptic digestion method to get peptide with 38 amino acids includes IgA1 hinge region. The HR glycopeptides with different compositions of O-glycans were monitored by multiple reaction monitoring. Product ion scan and glycosidase panel were used to confirm the analyte signals.
RESULTS: In total, 197 HR targets were included and the developed method was applied to human plasma samples from healthy control (n=4) and patients with IgAN (n=8). Different profiles of HR from each individual showed high diversity. It was previously reported that the levels of plasma IgA in patients with IgAN were higher than general concentrations. By using the simple tryptic digestion process, IgA1 representative peptide can also be monitored in the same run. In our system, plasma IgA1 can be determined from 0.087μg/μL to 5.62μg/μL. Responses of HR with different numbers of GalNAc (x), galactose (y), and sialic acid (z) were recorded. It was found that HR with 4 to 5 GalNAc and galactose showed higher responses among our study samples. Responses of IgA1 peptide and the HR glycopeptides were higher in IgAN group but the normalized responses showed similar patterns between groups.
CONCLUSION: The developed method has the potential to be used for personalized disease monitoring and investigating the IgA1 HR glycosylation patterns for IgAN. UHPLC-MS/MS with MRM detection mode is suitable to be set up in clinical chemistry lab.
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