Composition of Cell-Derived Matrices from Acomys cahirinus and Mus musculus - Label Free Quantitative Proteomics

The Spiny Mouse (Acomys) is gaining popularity as a research organism due to its phenomenal regenerative capabilities. However, access to Acomys colonies is limited and primary fibroblasts can only be maintained in culture for a limited time. To address these obstacles, we generated two immortalized Acomys dermal fibroblast lines. AcoSV40 fibroblasts were generated through lentiviral transfection with the SV40 Large T antigen. AcoSI fibroblasts were generated through spontaneous immortalization during prolonged subculture.

To demonstrate functional similarity between the two cell lines and primary Acomys fibroblasts (pAFs), we assessed deposited ECM proteins. Primary Mus fibroblasts and NIH3T3 fibroblasts were included as a comparison because NIH3T3 fibroblasts are a commonly used substitute for primary Mus fibroblasts. We made cell derived matrices (CDMs) with each of the 5 cell types by culturing them on collagen functionalized PDMS substrates for 7-28 days in media containing 25mg/mL Ficoll-400. Three sets of CDMs from each cell type were made. The CDMs were decellularized with a Triton X-100 and ammonium hydroxide solution, followed by a DNase treatment. Decellularized CDMs were then homogenized, acetone precipitated, and sent to the UF Mass Spectrometry Research and Education Center for label-free quantitative proteomics. Nano-liquid chromatography tandem mass spectrometry (Nano-LC/MS/MS) was performed on a Thermo Scientific Q Exactive HF Orbitrap mass spectrometer equipped with an EASY Spray nanospray source (Thermo Scientific) operated in positive ion mode. The LC system was an UltiMate™ 3000 RSLCnano system from Thermo Scientific. Precursor ion intensity label free quantitation was done using Proteome Discoverer (Thermo Fisher Scientific vs 2.4.0.305). The spreadsheets provided are the identified protein IDs found in each of the sample groups (Acomys, AcoSV40, AcoSI, Mus, NIH3T3). Three replicates are grouped into each result file.

Using these outputs, we performed the following comparisons : Acomys vs. AcoSV40, Acomys vs. AcoSI, and Mus vs. NIH3T3 in Proteome Discoverer. No significant differences in CDM composition were found between pAFs and AcoSV40 fibroblasts. AcoSI-1 CDMs shared 88% of proteins identified with pAFs. The proteins that differed between the two samples were involved in biological processes related to metabolism and translation, based on a GO enrichment analysis. In comparison, NIH3T3s, a commonly used substitute for Mus musculus cells, only shared 75% of proteins with their counterpart. Based on these results, AcoSV40 and AcoSI-1 should be representative of pAFs in experiments related to ECM deposition.