PRIDE Assigned Tags:Biomedical Dataset
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Proteomic characterization of stem cell-derived extracellular matrices
We characterized and compared the proteomic composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts isolated from different donors, employing a multidimensional proteomic approach coupled with label-based quantitative proteomics. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential proliferation and gene expression profiles between the cell types and as compared to TCPS, indicating that the cell-derived ECM influence each cell type in a unique manner.
Sample Processing Protocol
The ECM was mechanically detached from the 75 cm2-cell culture flask using a cell scraper in 2 ml of HBSS +/+, centrifuged at 16,000x g for 5 min, washed with 1 ml of HBSS +/+, centrifuged, and dried in a Speed-Vac (Savant) for 15 min. The ECM pellet was resuspended and reduced in a solution of 8 M urea, 100 mM ammonium bicarbonate, and 10 mM dithiothreitol at pH 8 under agitation at 37°C for 2 h. After cooling, cysteines were alkylated by adding iodoacetamide at a final concentration of 25 mM for 30 min. The ECM sample was then diluted to 2 M urea, 100 mM ammonium bicarbonate (pH 8), and deglycosylated with PNGaseF (2000 U, New England BioLabs, Ipswich, MA) for 2 h under agitation at 37°C, followed by digestion with Lys-C (Wako Chemicals USA, Richmond, VA), at a ratio of 1:100 enzyme:substrate, under agitation at 37°C for 2 h. Final digestion was done using trypsin (Sequencing Grade, Promega, Madison, WI), at a ratio of 1:50 enzyme:substrate, under agitation at 37°C overnight, followed by a second aliquot of trypsin, at a ratio of 1:100 enzyme:substrate, and an additional 2 h of incubation. Digests were acidified and desalted using 30mg HLB Oasis Cartridges (Waters Corp., Milford, MA) eluted with 50% acetonitrile with 0.1% trifluoroacetic acid (TFA), followed by concentration in a Speed-Vac.
Data Processing Protocol
Each sample was separated by reverse phase HPLC using an EASY-nLC1000 liquid chromatograph (Thermo Fisher Scientific, Waltham, MA) over a 140-minute gradient before nanoelectrospray using a Q Exactive mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in a data-dependent mode. The parameters for the full scan MS were: resolution of 70,000 across 350-2000 m/z; AGC 3e6; and maximum IT 50 ms. The full MS scan was followed by MS/MS for the top 10 precursor ions in each cycle with a NCE of 28 (unlabeled samples) or 32 (labeled samples) and dynamic exclusion of 30 s. Raw mass spectral data files (.raw) were searched using Proteome Discoverer (Thermo Fisher Scientific) and Mascot version 2.4.1 (Matrix Science) using the SwissProt Homo sapiens database containing 20,199 entries. Mascot search parameters were: 10 ppm mass tolerance for precursor ions; 0.8 Da for fragment ion mass tolerance; 2 missed cleavages of trypsin; fixed modification were carbamidomethylation of cysteines and for the quantitative experiments: TMT 6plex modification of lysines and peptide N-termini; variable modifications were oxidized methionines, deamidation of asparagines, pyro-glutamic acid modification at N-terminal glutamines; and hydroxylation of prolines and lysines.
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