Laser Microdissection and Mass Spectrometry: A Reliable Clinical Test for Detection of Membranous Nephropathy (MN) Antigens

Background: MN results from accumulation of antigen (Ag)-antibody (Ab) immune-complexes along the GBM. 13 target Ag have been discovered and include PLA2R, THSD7A, EXT1/EXT2, NELL1, SEMA3B, NCAM1, CNTN1, HTRA1, FAT1, PCDH7, NTNG1, PCSK6 and NDNF, accounting for ~80% of Ag. MN associated with many Ag have distinctive clinicopathologic finding and tissue/serum Ab have been described in all except EXT1/EXT2. As such, it is crucial to accurately identify the Ag in MN. Currently, IHC/IF methods are used to detect PLA2R, THSD7A, NELL1 and EXT1/EXT2. IHC/IF methods do not exist and are not practical for the remaining Ag. We have developed laser microdissection-mass spectrometry methodology (LMD/MS) as a one-stop clinical test for the detection of Ag using paraffin-embedded (FFPE) kidney biopsy tissue. Methods: For each case, 6 μM thick FFPE sections were mounted on Director slides and H&E stained. Using a Leica Laser Microdissection microscope, the glomeruli were dissected to reach ~250,000 μM2 per sample. Proteins were extracted using heat and sonication. Extracted proteins were reduced, alkylated and digested by incubating with trypsin. Resulting peptides were analyzed using an Exploris mass spectrometer connected to a liquid-chromatography system. Resulting MS/MS spectra were processed using three search engines to match the spectra against SwissProt human protein sequence database. Results: The LMD/MS test was validated in 2 steps: 1) LMD/MS was used to detect the Ag in 75 cases of MN with known Ag. LMD/MS correctly identified the Ag in all 75 cases (100%). 2) LMD/MS was used to identify the Ag in 61 MN cases where the Ag was unknown. LMD/MS identified one of the above Ag in 40 of 61 cases (65.5%) including less common Ag. In 21 cases (34.5%), none of the above Ag were detected. None of the negative controls were positive for any Ag. Conclusion: LMD/MS is an extremely useful and reliable method for the detection of MN Ag in the clinical laboratory.LMD/MS showing total spectral counts of MN Ag. Each column represents one MN case; 2 samples from each case are shown. Also shown in the bottom rows are housekeeping proteins actin and vimentin.