Deciphering the Kidney Matrisome: Identification and Quantification of Renal Extracellular Matrix Proteins in Healthy Mice

Abstract

Precise characterization of a tissue's extracellular matrix (ECM) protein composition (matrisome) is essential for biomedicine. However, ECM protein extraction that requires organ-specific optimization is still a major limiting factor in matrisome studies. In particular, the matrisome of mouse kidneys is still understudied, despite mouse models being crucial for renal research. Here, we comprehensively characterized the matrisome of kidneys in healthy C57BL/6 mice using two ECM extraction methods in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS), protein identification, and label-free quantification (LFQ) using MaxQuant. We identified 113 matrisome proteins, including 22 proteins that have not been previously listed in the Matrisome Database. Depending on the extraction approach, the core matrisome (structural proteins) comprised 45% or 73% of kidney ECM proteins, and was dominated by glycoproteins, followed by collagens and proteoglycans. Among matrisome-associated proteins, ECM regulators had the highest LFQ intensities, followed by ECM-affiliated proteins and secreted factors. The identified kidney ECM proteins were primarily involved in cellular, developmental and metabolic processes, as well as in molecular binding and regulation of catalytic and structural molecules' activity. We also performed in silico comparative analysis of the kidney matrisome composition in humans and mice based on publicly available data. These results contribute to the first reference database for the mouse renal matrisome.

Keywords: extracellular matrix; kidneys; label-free quantification (LFQ) of proteins; mass spectrometry; matrisome; mouse; protein identification; proteomics; tissue extraction.

Figure A1

Comparison of the methods of extraction of ECM proteins from mouse and human kidneys tissues.

Figure A2

The average composition of the matrisome revealed in mouse and human kidney samples by different studies, including the current study, and the following publications by McCabe et al. [20], Lipp et al. [28], Liu et al. [30], Louzao-Martinez et al. [31], and Randles et al. [32]. The bar graphs show Mean ± Standard Deviation of the number of proteins in each category averaged across the species-specific data reported in Table A9. Abbreviations: #—number of; GPs—ECM Glycoproteins; PGs—Proteoglycans.

Figure A3

Quantitative comparison of protein extraction levels between SME fractions and CME. Volcano plots represent significant differences in protein abundances (FC > 2, p-value < 0.05) between (a) SME (fraction 1) and CME, (b) SME (fraction 2) and CME and (c) SME (fraction 3) and CME. Top-right-hand sides of volcano plots are proteins that were significantly higher in SME fractions. Top-left-hand sides are proteins that were significantly higher in CME. Blue dots indicate Matrisome-associated proteins and red dots indicate core matrisome proteins that were significantly higher in either SME fractions or CME.

Figure A4

Quantified matrisome proteins in fractions of SME. Venn diagram illustrates the common and unique matrisome proteins in each fraction. Matrisome proteins were classified as collagens, ECM glycoproteins, proteoglycans, ECM-affiliated, ECM regulators and secreted factors.

Figure 1

Schematic illustration of the methods applied in the current study. (a) Matrisome protein extraction from healthy mouse kidneys by CME and SME methods. (b) Further processing and analysis of proteins after obtaining samples from methods CME and SME. Yellow color in the extraction products indicates the supernatant, and the orange color shows insoluble pellets. Abbreviations: SDS (Sodium Dodecyl Sulfate) and Gu-HCl (Guanidine Hydrochloride).

Figure 2

Identified matrisome proteins with unique peptides obtained from the samples processed via CME and SME methods. Venn diagram illustrates the common and unique matrisome proteins identified using each method. The proteins highlighted proteins in yellow have not been previously listed in the MD.

Figure 3

Mapping of the identified healthy mouse kidney matrisome proteins against GO terms using Uniprot database: (a) GO terms for the Biological Process and (b) GO terms for the Molecular Function.

Figure 4

Relative abundance of matrisome proteins in healthy mouse kidneys according to MaxQuant LFQ protein intensities. The results are presented separately for the products of CME and SME methods.

Figure 5

Heatmap of abundance of quantifiable matrisome proteins in the products of the CME and SME methods of sample preparation. Color coding in the heatmap depicts the variation between the maximum (coded in blue tones) to minimum (coded in red tones) observed LFQ intensity for each matrisome category and protein extraction method. The protein names shown by blue and red fonts are expressed above and below the average LFQ values in each matrisome category, respectively. The color coding scales (from blue for maximum LFQ intensity to red for minimum intensity per category) are provided on side of heatmaps (a–f).