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Abstract: FR-PO840

Quantitative Determination of Human IgA Subclasses in Plasma and Their Fc-Glycosylation Patterns by Using Peptide Analogue Internal Standard and an UHPLC-Triple Quadrupole Mass Spectrometry

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Kao, Chih-chin, Taipei medical university hospital, Taipei, Taiwan
  • Chen, Hsiao-Fan, Taipei Medical University, Taipei, Taiwan
  • Tsai, Isabel I-Lin, Taipei Medical University, Taipei, Taiwan
Background

Glycosylation on the Fab and Fc regions of immunoglobulins is important for immune function. In this study, we developed and validated a method for the quantification of immunoglobulin A by using affinity purification and UHPLC (ultra-high-performance liquid chromatography)-MS/MS. Twenty-seven IgA-related Fc N-glycopeptides were also detected.

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. After validation, the method was applied to plasma samples from 24 patients and 6 healthy controls, and the results were compared to those from ELISA assays.

Results

Correlation coefficients were greater than 0.999 for the IgA1 and IgA2 calibration curves and greater than 0.982 for glycopeptide regression curves. Intraday and interday precisions for IgA1 and IgA2 were <1.6% and <5.1% RSD, respectively. Intraday and interday accuracies ranged from 102.6-114.9% and 103.5-113.5% for IgA1 and IgA2, respectively. Recoveries for IgA1 and IgA2 in long-term and short-term stability ranged from 96.0-109.4%. Recoveries after three freeze-thaw cycles ranged from 93.2-113.2% for IgA1 and IgA2. The Pearson’s correlation was 0.84 when comparing the quantification of the 30 clinical samples by ELISAs and the developed UHPLC-MS/MS method.

Conclusion

IgA1 and IgA2 were isolated by peptide M purification. An efficient on-bead digestion process was used prior to UHPLC-MS/MS analysis. The validated method was successfully applied to clinical samples which showed high correlation to the total IgA quantification ELISA results. This method has potential for investigating IgA profiles from human plasma.

Figure 1. Workflow of IgA purification and UHPLC-MS/MS analysis.