MSACL 2019 EU Abstract

Quantitative Determination of Human IgA Subclasses and their Fc-Glycosylation Patterns in Human Plasma by Using Peptide Analogue Internal Standard and UHPLC-MS/MS

Hsiao-Fan Chen(1), Chih-Chi Kao(2, *), I-Lin Tsai(3, *)
(1)Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (2)Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, College of Pharmacy, Taipei Medical University, Taipei, Taiwan (2) International PhD Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (3) Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

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Hsiao-Fan Chen (Presenter) Taipei Medical University >> POSTER (PDF)

Presenter Bio: Hsiao-Fan Chen is a graduate student from Taipei medical university, major in nutrition and health science from 2015-2019. At the same time, she is a pre-master student of graduate institute of medical science, college of medicine, Taipei medical university in her fourth year of college life wishing to spend her time more efficiently. She joined professor I-Lin Tsai’s laboratory in the second semester in sophomore. She has been learning analytical chemistry for almost half and two years. During that time, she has learned the techniques of SDS-PAGE, ELISA and sample preparation for LC/MS analyze. Currently, she is working on biomarker investigation of an autoimmune disease using UHPLC-MS/MS.
Prior to the project, she has learned about how to read the meanings of chromatograms of analytes. First, they were working on Chinese medicine analysis in a HPLC-MS/MS system. Since then, she was get

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Rationale:
Glycosylation on the Fab and Fc regions of immunoglobulins is important for our immune function. In this study, we developed and validated a method for quantification of immunoglobulin A by using affinity purification and UHPLC-MS/MS analysis. Twenty-seven of IgA related Fc N-glcopeptides were also detected simultaneously.
Methods:
Peptide M was used to purify IgA, and a peptide analog was added as an internal standard. After on-bead digestion, samples were analyzed by UHPLC-MS/MS instrument. After validation, the method was applied to plasma samples from 24 patients and 6 healthy controls. And the results were compared to ELISA assay.
Results:
Correlation coefficient was higher than 0.999 in IgA1 and IgA2 calibration curves and over than 0.979 in regression curves of glycopeptides. Intra-day and inter-day precisions for IgA1 and IgA2 were <1.6% and <5.1% RSD. Intra-day and inter-day accuracies were ranged from 102.6-114.9% and 103.5-113.5% for IgA1 and IgA2. Recoveries for IgA1 and IgA2 in long term and short term stability were ranged from 96.0-109.4%. Recoveries after freeze and thaw for three cycles were ranged from 93.2-113.2% for IgA1 and IgA2. The Pearson’s correlation is 0.84 for the quantification results of 30 clinical samples by using ELISA and the developed UHPLC-MS/MS method.
Conclusions:
Purification of IgA1 and IgA2 was developed by using peptide M purification. An efficient on-bead digestion process was used before UHPLC-MS/MS analysis. The validated method was successfully applied to clinical samples which showed high correlation to the total IgA quantification results from ELISA. This method has the potential for investigating IgA profiles from human plasma.