Mass Spectrometry With Data-Independent Acquisition for the Identification of Target Antigens in Membranous Nephropathy

In recent years, the strategy of using laser microdissection and mass spectrometry (LCM/MS) has expanded the landscape of antigens associated with membranous nephropathy (MN). Specific associations with phenotypes, diseases and sometimes reversible triggers led to an antigen-based classification of MN, informing precision medicine and highlighting for the potential value of routine use of proteomics in classifying MN. This study aimed at reproducing and further improving the original LCM/MS for antigen detection in MN. Retrospective cohort study using residual material from kidney biopsies. We applied LCM/MS to kidney biopsy specimens from 64 individuals, including 31 healthy controls; 5 PLA2R-associated MN; 23 PLA2R-negative MN; and 5 individuals without disease. Proteomic analysis of microdissected glomeruli. Protein abundance and C3 fragments in PLA2R-MN vs. controls; identification of target antigens in PLA2R-negative MN. The technique of LCM/MS was expanded by integrating a data-independent acquisition (DIA) approach to enable the identification and quantification of peptides of varying abundance. We observed significant enrichment in PLA2R, IgG4, and complement proteins, providing molecular evidence for complement activation in glomeruli from patients with PLA2R-MN. Compared to conventional DDA, DIA increased the number of glomerular proteins (∼3800 vs. ∼1200) identified in healthy glomeruli; allowed the detection of all known antigens except NELL1 in normal glomeruli; and increased the detection rate of antigens from 46% to 83% in PLA2R-negative MN. Retrospective design; sample size; no identification of novel antigens. An integrative approach combining LCM/MS and DIA enabled identification of more target antigens than LCM/MS with DDA, potentially informing the understanding of disease mechanisms in MN.