Composition of Cell-Derived Matrices from Acomys cahirinus and Mus musculus - Label Free Quantitative Proteomics
Description
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.
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CDMs were obtained by seeding pAFs, AcoSV40, or AcoSI-1 fibroblasts onto collagen functionalized Sylgard 527 PDMS. Each substrate was plasma treated to oxidize the surface before functionalizing with (3-Aminopropyl)trimethoxysilane, followed by glutaraldehyde and then rat tail collagen I. Functionalization with collagen I is needed to prolong adherence of Acomys cells to the substrate. Cells were cultured for 7-28 days in media containing 25mg/mL Ficoll 400, with immortalized cells requiring less time in culture to generate similar protein amounts as pAFs. Following this period, CDMs were decellularized by treating with a solution of 0.5%Triton X-100 and 0.3M ammonium hydroxide in PBS for 5 minutes. The presence of residual DNA was reduced by treating decellularized CDMs with 10ug/mL DNase I at 37oC for 30 minutes. Decellularized CDMs were homogenized with RIPA Buffer and a tissue homogenizer. The samples were at 3200g and 4oC for 15 minutes, followed by 14,000g for 2 minutes. The supernatant was removed and stored at -20oC as the RIPA soluble protein fraction. The pellet was resuspended in a membrane solubilization buffer containing 40mM Tris-Cl (pH 8.0), 7M urea, 2M thiourea, 0.25% w/v ASB-14, and 0.25% NP-40 and incubated at room temperature for 15 minutes. The samples were vortexed for 1 minute, then centrifuged at 3200g for 30 minutes. The supernatant was removed and diluted by a factor of 2.5 with dH2O and stored at –20oC as the RIPA insoluble protein fraction. Prior to analysis, both the RIPA soluble and RIPA insoluble protein fractions were precipitated in ice-cold acetone overnight. The solutions were centrifuged at 4oC and 3200g for 15 minutes. The acetone was fully removed, and the remaining pellets were resuspended in 2M urea. Samples were sent to the UF Mass Spectrometry Research and Education Center for label-free quantitative proteomics. The scan sequence of the mass spectrometer was based on the original TopTen™ method; the analysis was programmed for a full scan recorded between 375 – 1575 Da at 60,000 resolution, and a MS/MS scan at resolution 15,000 to generate product ion spectra to determine amino acid sequence in consecutive instrument scans of the fifteen most abundant peaks in the spectrum. The AGC Target ion number was set at 3e6 ions for full scan and 2e5 ions for MS2 mode. Precursor ion intensity label free quantitation was done using Proteome Discoverer (Thermo Fisher Scientific vs 2.4.0.305). The two groups (B33p4 vs Hp4) were compared using a “non-nested” study factor. Normalization was derived by using all peptides. Protein abundances were calculated by summed abundances, meaning the protein abundances are calculated by summing sample abundances of the connected peptide groups. Fisher’s exact test (pairwise ratio-based) was used to calculate p-values with no missing value imputation included. Adjusted p-values were calculated using Benjamini-Hochberg.