# Benefits and limits of decellularization on mass-spectrometry-based extracellular matrix proteome analysis of mouse kidney

Teresa Frattini, Hanne Devos, Manousos Makridakis, Maria Roubelakis, Agnieszka Latosinska, Harald Mischak, Joost Schanstra, Antonia Vlahou, Jean-sébastien Saulnier-Blache

► To cite this version:

Teresa Frattini, Hanne Devos, Manousos Makridakis, Maria Roubelakis, Agnieszka Latosinska, et al. Benefits and limits of decellularization on mass-spectrometry-based extracellular matrix proteome analysis of mouse kidney. Proteomics, 2024, 24 (17), pp.e2400052. 10.1002/pmic.202400052. hal-04920968

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## DATASET BRIEF

# Benefits and limits of decellularization on mass-spectrometry-based extracellular matrix proteome analysis of mouse kidney

Teresa Frattini<fnref n="1" />,<fnref n="2" />

Hanne Devos<fnref n="3" />

Manousos Makridakis<fnref n="3" />

Maria G. Roubelakis<fnref n="4" />,<fnref n="5" />

Agnieszka Latosinska<fnref n="6" /> ID

Harald Mischak<fnref n="6" /> ID

Joost P. Schanstra<fnref n="1" />,<fnref n="2" /> ID

Antonia Vlahou<fnref n="3" /> ID

Jean-Sébastien Saulnier-Blache<fnref n="1" />,<fnref n="2" /> ID

### Correspondence

Antonia Vlahou, Biotechnology Division,
Biomedical Research Foundation, Academy of
Athens, Soranou Efessiou 4, Athens 11527,
Greece.

Email: vlahoua@bioacademy.gr

Jean-Sébastien Saulnier-Blache, Institut
National de la Santé et de la Recherche
Médicale, U1297, Institute of Cardiovascular
and Metabolic Disease, Toulouse, France.

Email:
jean-sebastien.saulnier-blache@inserm.fr

### Funding information

Agence Nationale de la Recherche,
Grant/Award Number: ANR-21-CE18-0031;
HORIZON EUROPE Marie Sklodowska-Curie
Actions, Grant/Award Number: 101072828

### Abstract

The extracellular matrix (ECM) is composed of collagens, ECM glycoproteins, and proteoglycans (also named core matrisome proteins) that are critical for tissue structure and function, and matrisome-associated proteins that balance the production and degradation of the ECM proteins. The identification and quantification of core matrisome proteins using mass spectrometry is often hindered by their low abundance and their propensity to form macromolecular insoluble structures. In this study, we aimed to investigate the added value of decellularization in identifying and quantifying core matrisome proteins in mouse kidney. The decellularization strategy combined freeze-thaw cycles and sodium dodecyl sulphate treatment. We found that decellularization preserved 95% of the core matrisome proteins detected in non-decellularized kidney and revealed few additional ones. Decellularization also led to an average of 59 times enrichment of 96% of the core matrisome proteins as the result of the successful removal of cellular and matrisome-associated proteins. However, the enrichment varied greatly among core matrisome proteins, resulting in a misrepresentation of the native ECM composition in decellularized kidney. This should be brought to the attention of the matrisome research community, as it highlights the need for caution when interpreting proteomic data obtained from a decellularized organ.

<figure><img src="image_2.png" /></figure>

<footnote n="1">¹Institut National de la Santé et de la Recherche Médicale, U1297, Institute of Cardiovascular and Metabolic Disease, Toulouse, France</footnote>

<footnote n="2">²Université Toulouse III Paul-Sabatier, Toulouse, France</footnote>

<footnote n="3">³Biotechnology Division, Biomedical Research Foundation, Academy of Athens, Soranou Efessiou 4, Athens, Greece</footnote>

<footnote n="4">⁴Laboratory of Cell and Gene Therapy, Biomedical Research Foundation, Academy of Athens, Athens, Greece</footnote>

<footnote n="5">⁵Laboratory of Biology, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece</footnote>

<footnote n="6">⁶Mosaiques Diagnostics GmbH, Hannover, Germany</footnote>

<footnote>Abbreviations: ND, non-decellularized; D, decellularized; FTC, freeze-thaw cycles.</footnote>

<footnote>T. Frattini and H. Devos share co-first authorship with equal contribution.</footnote>

<footnote>This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2024 The Author(s). *PROTEOMICS* published by Wiley-VCH GmbH.</footnote>
## KEYWORDS

decellularization, extracellular matrix, kidney, matrisome

The extracellular matrix (ECM) is crucial to regulate cell and tissue structure and function, and its dysregulation is associated with a wide variety of diseases including fibrosis [1, 2]. Within the ECM, two main components can be distinguished: the core matrisome corresponding to ECM proteins per se (collagens, ECM glycoproteins, proteoglycans) and the matrisome-associated proteins (ECM-affiliated proteins, ECM regulators, secreted factors). Matrisome-associated proteins maintain the core matrisome proteins by balancing their production and degradation [3]. This balance is of vital importance, as abnormal ECM remodelling drives the deregulation of cell proliferation, the loss of cell differentiation and the failure of cell death, resulting in disease progression and pathological processes including tissue fibrosis [4].

Mass-spectrometry-based proteome analysis offers a valuable, unbiased method to explore ECM components [5, 6]. However, this is hindered by several factors. Firstly, ECM proteins represent frequently roughly 1% of the tissue proteome and could be masked by the 99% remaining cellular proteins, depending on the organ and the proteomic method used [7-9]. In addition, core matrisome proteins tend to create insoluble macromolecules in the aqueous solutions used for mass spectrometry sample preparation. Moreover, ECM proteins undergo post-translational modifications, such as proline and lysine hydroxylation in the case of collagens [7-9], which increase their proteomic complexity. Taken together, these aspects make optimal ECM protein detection and identification a complicated task.

Decellularization can produce enriched-ECM samples suitable for proteome analysis by removing intracellular and ECM-associated proteins otherwise masking ECM components [10-12]. Decellularization strategies exploit chemical (e.g., detergents, alcohols), physical (e.g., temperature, force and pressure) and biological treatments (e.g., enzymes) to eliminate intracellular and matrisome-associated proteins, while preserving core matrisome proteins. Decellularization is often used in tissue engineering and regenerative medicine to create biological scaffold for reconstructive surgical application [13]. At the downside, proteins in decellularized samples might not fully capture the complete core matrisome, due to accidental losses of ECM proteins during sample preparation and/or the possible presence of residual intracellular proteins interfering with the analysis [14, 15]. Further challenges, such as the solubilization of macromolecules and the identification of post-translational modifications on the ECM proteins remain [14, 16].

Our objective was to compare the core matrisome, focussing on collagens, of decellularized (D) versus non-decellularized (ND) samples in mouse kidney using mass spectrometry-based proteomics (Study workflow summarized in Figure S1). Kidney was chosen as a use-case, because kidney fibrosis is the final pathological manifestation of many chronic kidney diseases (CKD) [17] and a major cause of morbidity and mortality [18]. The literature reports several ECM-proteome pro-

files of decellularized kidney [19-22] but, to our knowledge none of them were compared with the native non-decellularized kidney. Here we report a comparison of the ECM proteome between decellularized and non-decellularized mouse kidney. The decellularized protocol that we used (Supplementary Methods) was adapted from previously published reports [13, 23, 24], and combined freeze-thaw cycles (FTCs) with Sodium Dodecyl Sulphate (SDS) treatments. The merge of the two treatments was based on the evidence that, compared to decellularization by chemical treatments alone, previous freeze-thawing cycles showed the most effective result in reducing DNA while also preserving glycosaminoglycan (GAG) content [23]. SDS was reported as the most appropriate detergent to identify ECM proteins using mass spectrometry [24] and to preserve the natural architecture of the tissue [25, 26], although it was reported to alter hydrogel cytocompatibility [27]. Since our objective was to identify ECM proteins in decellularized samples and not to analyse hydrogel functionality, we decided to choose SDS for our protocol.

We first assessed the influence of decellularization on kidney histology. D and ND kidney samples were stained either with Haematoxylin and Eosin (H&E) to visualize the whole tissue structure including cell nuclei, or with Masson's Trichrome to visualize collagen fibres (Supplementary Methods). ND samples exhibited a typical tubulo-glomerular organisation with clearly visible cell nuclei (purple dots, black arrows) (Figure S2A, C), with occasionally the presence of local trabeculae of collagen fibres stained (blue staining in Figure S2C). In D samples, the tubulo-glomerular organisation and the cell nuclei were lost (Figure S2B), and most of the surface was occupied by collagen (stained in blue) (Figure S2D), suggesting efficient decellularization while preserving collagen structures.

We then performed LC-MS/MS-based proteome analysis (see Supplementary Methods) to identify and quantify the proteins present in D (3 biological replicates) and ND (2 biological replicates) kidney samples. Among a total of 2241 proteins detected at least once across the five samples (Table S3), 1263 (56.4%) proteins were in common to D and ND samples, 57 (2.5%) were detected only in the D samples, and 921 (41.1%) were detected only in the ND samples (Figure S3). The total number of the detected proteins (Figure S4A) and their abundance distribution (Figure S4B) were consistent throughout the samples within each group. With reference to matrisomeDB database (https://matrisomedb.org) (details in Supplementary Methods), 72 proteins were annotated as core matrisome, 63 proteins as matrisome-associated and 2106 proteins as cellular. The total number of detected proteins was reduced by 48% in D compared to ND samples (Figure 1A). This was mainly due to a reduction in the number of cellular (41.5%) and matrisome-associated proteins (23.6%). In contrast, a very large proportion (95%) of core matrisome proteins detected in ND samples were also observed in D samples, and was even increased
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**FIGURE 1** Protein number. (A) Proteins detected at least once in ND and D samples were annotated as Core matrisome or Matrisome-associated with reference to matrisomeDB data base. The proteins not annotated in matrisomeDB were labelled Cellular. (B) Core matrisome and Matrisome-associated protein subcategories. Abbreviations: ND, non-decellularized; D, decellularized.</figcaption></figure>

(Figure 1B) with the detection of two additional collagens, nine additional ECM-glycoproteins and one additional proteoglycan (Table S4). The fact that their average abundance remained much lower (130-fold) compared to the other core matrisome proteins may explain why these additional proteins were not detected in the native ND kidney (Table S3). Notably, three core matrisome proteins (Biglycan, Decorin, and von Willebrand factor A domain-containing protein 5A) were lost. Biglycan and Decorin are glycoaminoglycans, a class of molecules known to be completely solubilized by SDS [28]. In addition, while some glycoaminoglycans are associated with the ECM scaffold, most of them are cell-associated [29]. This might explain why they were eliminated by decellularization. Von Willebrand factor A domain-containing protein 5A is a soluble nucleoplasmic protein [30] that might have been eliminated together with nuclei. Taken together, above observations indicated that decellularisation preserved the vast majority of core matrisome proteins detected in native kidney, while removing a significant number of cellular and matrisome-associated proteins. This agrees with the resistance of core matrisome protein extraction to SDS extraction, in contrast to most intracellular and matrisome-associated proteins anchored in the ECM.

The impact of decellularization was even more striking when analysing protein abundance. Considering the proteins detected at least once in each group, the sum of abundance of the cellular proteins represented more than 97% of the total in the ND samples, dropping to 22.6% in the D samples (Figure 2A, Table S3). In contrast, the sum of abundances of core matrisome proteins that represented 1.3% of total in ND samples, increased to 76.7% in D samples (Figure 2A). The proportion of matrisome-associated proteins remained low in both ND and D samples (1.2 and 0.5%, respectively) (Figure 2A). Considering the proteins shared by all samples (D and ND), 80.1% were depleted, and 19.8% were enriched by decellularization (Figure 2B, Table S3). Depletion concerned 85.3% of cellular proteins and 78.3% of matrisome-associated proteins (Figure 2B). In contrast, enrichment concerned 96.1% of the Core matrisome proteins (100% of Collagens and 75% of Proteoglycans (Figure 2B)). Average enrichment of core matrisome proteins was 59.1-fold, with collagens, ECM-glycoprotein and proteoglycans being enriched by 99.9, 43.2 and 12.8-fold, respectively (Figure 2B). Only two core matrisome proteins (Lumican and Insulin-like growth factor-binding protein complex acid labile subunit) showed a ratio <1 (Table S3). Enrichment of core matrisome proteins and depletion of cellular proteins and matrisome-associated proteins are also evidenced when plotting ND to D individual proteins abundance (Figures 2D and S5). Furthermore, abundances of the individual core matrisome proteins in ND and D samples were significantly correlated (Spearman rho 0.84, p<0.0001) (Figure 3A). When examining protein abundances in more details, enrichment varied extensively from one protein to another (Table S3). As an example, enrichment of collagens varied from 14- to 287-fold after decellularization (Figure 3B). Therefore, while decellularisation allowed recovery and enrichment of the vast majority of core matrisome proteins, their enrichment was not consistent from one protein to another. This constitutes a drawback of decellularization since it introduces a biased picture of the native organ composition in ECM proteins.

A limitation of this study was that sample size was too small to allow comparative statistical analyses between D and ND samples. However, our current data were already fairly reproducible (e.g., ID overlap of 98.5% and coefficient of variation of 20.5 ± 1.99% for the core matrisome proteins throughout the 3D samples). However, future research studies are needed to validate these discoveries using larger samples size.

In conclusion, although decellularization strongly enriched the core matrisome proteins, their unequal enrichment represents a major drawback of this strategy. This should be brought to the attention of the matrisome research community, as it highlights the need for caution when interpreting proteomic data obtained following a
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**FIGURE 2** Protein abundance. (A) Sum of abundance of the proteins detected at least once each group. Values are means ± SD. (B) D/ND abundance ratio distribution of detected proteins shared by D and ND samples. Abbreviations: ND, non-decellularized; D, decellularized.</figcaption></figure>

decellularization procedure. To obtain a full image of the core matrixome composition of a given tissue, analysis of the decellularized samples should go along with the analysis of the decellularized fraction even if this comes at the cost of concentrating proteins from large

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**FIGURE 3** (A) Mean abundance plot of individual Core matrisome proteins between ND to D. Spearman correlation analysis: 0.8361 (*p* < 0.0001), -0.0036 (*p* = 0.9857), and 0.4600 (*p* < 0.0001). (B) D/ND abundance ratio of individual Collagens. Abbreviations: ND, non-decellularized; D, decellularized.</figcaption></figure>

volumes of washes and of doubling the number of proteomic analysis runs.

## AUTHOR CONTRIBUTIONS

T. Frattini, H. Devos and M. Makridakis performed the experiments and data analysis, and wrote the initial version of the manuscript. A. Latosinska and H. Mischak performed LC-MS/MS and data analysis, and provided critical feedback and helped shape the analysis and manuscript. M. Makridakis and M. G. Roubelakis contributed to the interpretation of the results and provided critical feedback and helped shape the analysis and manuscript. J. P. Schanstra, A. Vlahou and J.-S. Saulnier-Blache designed the study and were in charge of overall direction, planning and final manuscript writing.

## ACKNOWLEDGEMENTS

The authors were supported by the European Union's Horizon 2020 EU-Cost PerMedik action (CA21165). A Vlahou, H Mischak and JP Schanstra are members of Eutox. The present work was funded in the
frame of the « DisCo-I » project that has received funding from the European Union's Horizon Europe Marie Skłodowska-Curie Actions Doctoral Networks - Industrial Doctorates Programme (HORIZON - MSCA - 2021 - DN-ID) under grant agreement No 101072828 and by the Agence Nationale de la Recherche (ANR) Treat-CKD project ANR-21-CE18-0031.

## CONFLICT OF INTEREST STATEMENT

H.M. is the co-founder and co-owner of Mosaiques Diagnostics. A.L. is employed by Mosaiques Diagnostics. All other authors declare no conflict of interest. All other authors declare no conflict of interest.

## DATA AVAILABILITY STATEMENT

Raw data files (Table S5) have been deposited on the MassIVE (Mass Spectrometry Interactive Virtual Environment) repository (identifier: MSV000094031) and on ProteomeXchange repository (identifier: PXD049247).

## DISCLAIMER

Funded in part by the European Union. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.

## ORCID

Agnieszka Latosinska https://orcid.org/0000-0001-8917-2412

Harald Mischak https://orcid.org/0000-0003-0323-0306

Joost P. Schanstra https://orcid.org/0000-0002-7471-372X

Antonia Vlahou https://orcid.org/0000-0003-3284-5713

Jean-Sébastien Saulnier-Blache https://orcid.org/0000-0002-8971-7723

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## SUPPORTING INFORMATION

Additional supporting information may be found online at https://doi.org/10.1002/pmic.202400052 in the Supporting Information section at the end of the article.

**How to cite this article:** Frattini, T., Devos, H., Makridakis, M., Roubelakis, M. G., Latosinska, A., Mischak, H., Schanstra, J. P., Vlahou, A., & Saulnier-Blache, J.-S. (2024). Benefits and limits of decellularization on mass-spectrometry-based extracellular matrix proteome analysis of mouse kidney. *Proteomics, 24*, e2400052. https://doi.org/10.1002/pmic.202400052