Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

Identification and quantification of matrisome proteins in mouse kidneys (#172)

Umut Mr Rende 1 , Charlie Dr Ahn 2 , Subash Dr Adhikari 3 , Anna Dr Guller 1 2
  1. University of New South Wales, Sydney, NEW SOUTH WALES, Australia
  2. Macquarie University, Sydney
  3. The Walter and Eliza Hall Institute of Medical Research, Melbourne

Excessive accumulation of extracellular matrix (ECM) is a hallmark of fibrotic diseases. Understanding the matrisome (ECM proteome) composition, homeostasis and mechanisms of physiological versus pathological response to injury is essential for the discovery of anti-fibrotic drugs and diagnostic tools. The challenging obstacle in studying the matrisome is the need for optimization of matrisome enrichment methods for different organs, diseases, and species. Currently, there is no available optimized protocol, nor a publicly available matrisome database for mouse kidneys. This limits the power of murine models in renal fibrosis research.

In this study, we aimed to find the optimal matrisome extraction method applicable to mouse renal tissues using a comparative analysis of the Millipore Compartment Fractionation (Method-1)(Naba et al, 2015, J.Vis.Exp) and the Sequential Extraction (Method-2)(Barallobre-Barreiro et al, 2017, J.Vis.Exp) approaches. We examined the efficiency of these methods in matrisome profiling of healthy C57BL/6 mice kidneys (n=3 per method) by LC-MS/MS, peak identification and label-free quantification using MaxQuant.

Overall, we identified 120 matrisome proteins (MPs), including 24 proteins that have not been previously listed in MatrisomeDB (http://matrisomeproject.mit.edu). By Methods-1 and -2, respectively, 72/120 (60%) MPs and 118/120 (98.3%) MPs were detected, and 48/72 (66.7%) and 85/118 (72%) MPs quantified. Among the MPs quantified by Method-1, 29 and 19 were core matrisome and ECM-associated proteins, respectively. By Method-2 we did not only identify all core MPs (except Fibrinogen α-chain) and ECM-associated proteins that were revealed by Method-1 but additionally quantified 7 core matrisome and 31 ECM-associated proteins.

In conclusion, matrisome enrichment by the sequential extraction allows to identify and quantify more MPs in healthy murine kidneys. Our results can contribute to the further studies of kidney ECM turnover in normal and pathological conditions and the identification of novel drug targets and biomarkers for the treatment and diagnostics of chronic kidney diseases.