Multiplexed Detection of Membranous Nephropathy Antigens by Multi-Reaction Monitoring Mass Spectrometry

Membranous nephropathy (MN) is a leading cause of nephrotic syndrome, driven by an immune response against a target autoantigen. To date, over 30 target antigens have been described in MN, creating the need for multiplex approaches. We developed a multiple reaction monitoring (MRM)-based assay for the clinical mass spectrometry (MS) laboratory for determination of an antigen type in nephropathology practice. Here, we provide detailed methods and validation data to ensure adaptability among different instruments and laboratories.

Protein A/G immunoprecipitation was used to extract immune complexes from frozen kidney biopsy tissue. Proteins were eluted from magnetic beads, reduced, alkylated, and trypsin digested using single-pot, solid-phase-enhanced sample preparation workflow. Tryptic peptide quantities were measured by MS with internal standard (IS) peptides. Peak area ratios were used to call an antigen type. Immunostaining was used as an independent method to confirm target antigen types. A calling algorithm was empirically developed that maximizes sensitivity and specificity of the workflow for identifying membranous antigens in kidney biopsies.

Our immunoprecipitation-to-MS workflow demonstrated reliable quantitative detection of a total of 19 MN antigens. The MS-enabled workflow had a diagnostic accuracy of 97.2% with a sensitivity of 97.2% and 100% specificity from a validation cohort of 113 MN cases with antigen type confirmed by immunostaining.

MRM-MS following protein A/G immunoprecipitation of MN biopsy tissue can be effectively used for multiplex antigen typing in clinical nephropathology practice.