MSACL 2019 EU Abstract

Incorporating Stable Isotope-Labeled IgG Internal Standard and Affinity Purification for Analyzing Human IgG4 and Fc-glycan Profiles by UHPLC-MS/MS

Jing-Ya Shiao (1), Yu-Ting Chang (2,3), Ming-Chu Chang (2,3), Isabel I-Lin Tsai (1,4,5,6,7, *)
(1)Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University (2) Department of Internal Medicine, National Taiwan University Hospital (3) Department of Internal Medicine, College of Medicine, National Taiwan University (4) Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan (5) Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, School of Pharmacy, Taipei Medical University, Taipei, Taiwan (6) International Ph.D. Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (7) Pulmonary Research Center, Wan Fang Hospital, Taipei Medical University

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Isabel I-Lin Tsai (Presenter) Taipei Medical University >> POSTER (PDF)

Relevant Financial Disclosures (within past 24 months) Grant/Research Support Ministry of Scinece and Technology Conflict mitigation NOT REQUIRED.

INTRODUCTION:
It is reported that IgG Fc-glycosylation is distinct between patients with autoimmune pancreatitis (AIP) and pancreatic ductal adenocarcinoma (PDAC), which has the potential to be used to benefit differential diagnosis in clinical application.
OBJECTIVES:
Since type I AIP is categorized as IgG4 related disease (IgG4-RD), we aimed to purify IgG4 from human serum and focused on analyzing the Fc-glycosylation profiles. The purification of IgG4 can also solve the issue of isobaric Fc-glycopeptides from human IgG3 and IgG4 of Asia population.
METHODS:
IgG4 affinity beads were used to purify IgG4 from 10 μL of human serum. The incubation time for purification is 0.5 hr, and the on-bead digestion at 37℃ was conducted for 16 hr to get IgG4 representative peptide and glycopeptides from the Fc region. Stable isotope-labeled IgG internal standard (IS) was added before affinity purification which can correct the variation of purification and tryptic digestion efficiently. The targets were analyzed by using UHPLC-MS/MS with multiple reaction monitoring (MRM) mode.
RESULTS:
The concentration range for calibration curve was 0.14-8.8 μg/μL with an r-value of 0.999. The inter-day and intra-day precision of peak area ratios of IgG4 peptide to IS were less than 4.8 % RSD for LLOQ, low, medium and high QC samples. The inter-day and intra-day accuracies were between 95.7-101.8 % for the 4 concentration levels. Matrix effects for low, medium and high QC samples were 105.8-118.9 %. The target peptide was stable in the matrix (chicken serum) for 15 days in -80 ℃ and the recoveries for three freeze-thaw cycles were 85.3-93.7 %. The prepared samples were also stable in autosampler for 24 hr with the temperature set at 4 ℃. The developed and validated method was also applied to clinical samples from patients with AIP and PDAC, and the profiles were compared in this study.
CONCLUSION:
IgG4 quantification and the monitoring of Fc-glycan profiles can be achieved efficiently by using UHPLC-MS/MS with MRM mode. And the developed workflow is suitable for clinical applications.