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21746 Antigen discovery in membranous glomerulopathy using laser capture microdissection and mass spectrometry

Published online by Cambridge University Press:  30 March 2021

Jacquelyn Fede
Affiliation:
University of Rhode Island
Stephen Kogut
Affiliation:
University of Rhode Island
Anthony Heyward
Affiliation:
Warren Alpert Medical School, Brown University
John F. Stevenson
Affiliation:
University of Rhode Island
Amy Nunn
Affiliation:
Brown University
Julie Plaut
Affiliation:
Brown University
Judy A. Kimberly
Affiliation:
Brown University
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Abstract

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ABSTRACT IMPACT: Identifying the causative antigen in membranous glomerulopathy cohorts enables the development of serum assays to detect and monitor disease progression without the need for invasive kidney biopsies. OBJECTIVES/GOALS: Primary membranous glomerulopathy is caused by the formation of autoantibody immune complexes which deposit in the glomerulus and obstruct kidney function. Causative antigens remain to be identified in roughly 20% of cases. Our goal is to identify the antigen in these cohorts, so that non-invasive assays can be developed for disease monitoring. METHODS/STUDY POPULATION: Renal biopsy tissue from known antigen cases (PLA2R, THSD7A), and unknown cases were included in the analysis. Renal biopsy tissue from formalin fixed paraffin embedded tissue was cut at a thickness of 10 µm onto Leica PET-membrane frame slides. These slides were then stained with hematoxylin. The glomeruli were microdissected into microcentrifuge tubes using a Leica DM6000B microscope. The microdissected glomeruli were lysed in 2% SDS and 0.1M DTT at 99 degrees Celsius for 1 hour and processed by filter assisted sample preparation (FASP). Digested peptides were analyzed by liquid chromatography-mass spectrometry using an Orbitrap Fusion Lumos using data-dependent acquisition. RESULTS/ANTICIPATED RESULTS: Mass spectrometry data collected from the laser captured glomeruli was searched against the human proteome fasta database from Uniprot using MaxQuant. IBAQ values were used for quantitation and statistical analysis. Null hypothesis significance testing was performed for each protein by comparing each sample group to the rest of the samples in the data set. In the control groups, the causative antigens PLA2R and THSD7A were detected and quantified with the largest magnitude fold change in their respective category, validating the experimental design. Using this approach, the proteins SAP, NELL1, and NCAM1 were identified and subsequently validated as causative antigens in distinct patient cohorts. DISCUSSION/SIGNIFICANCE OF FINDINGS: Here, we share the results of our efforts to comprehensively identify the spectrum of causative antigens in membranous glomerulopathy. In this context, antigen discovery is an essential first step for the development of non-invasive assays to inform prognosis, monitor response to treatment, and better understand disease etiology.

Type
Basic Science
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright
© The Association for Clinical and Translational Science 2021