Figure 1.
Flow chart of ECM preparation, solubilization and analysis.
Table 1.
Proteins detected in SB623-derived ECM using In-Gel Digest and nLC-MS/MS or SAISD and nLC-MS/MS.
Table 2.
Percentage of total spectral counts for SB623- and MSC-derived ECM proteins identified using SAISD and LC-MS/MS.
Figure 2..
Isolation and relative protein expression comparison of MSC and SB623-derived ECM.
SB623 and MSCs were cultured for five days in complete medium followed by two days in serum-free medium (A). Cells were treated with 0.2% Triton X-100 (Triton) (B). Cells were then treated with 0.3% NH4OH, DNase I and PMSF, and then rinsed. The isolated ECM has a fibril-matrix like appearance (C). MSCs and SB623 produce similar quantities of ECM proteins. ECM protein concentrations were determined using the BCA assay, and relative ECM protein expression was determined by normalizing to relative cell counts (LDH assay) (D). Scale bar = 50 µm.
Figure 3.
Solubilized MSC and SB623-derived ECM analyzed by SDS-PAGE. SDS/urea-soluble SB623-derived ECM (SB) and corresponding MSC.
M: molecular weight markers. SDS/urea samples were precipitated, re-suspended in 2X loading buffer, loaded on a 1.5-mm 4–20% Tris-acetate gel, electrophoresed and stained with Coomassie Blue R-250.
Figure 4.
Proteins that exhibit differential expression between SB623- and MSC-derived ECM; either in SDS/urea-soluble or SDS/urea-insoluble fractions or both.
Precipitated ECM samples were resuspended in 0.1% w/v ammonium bicarbonate and Rapigest surfactant powder. The sample was then reduced, alkylated, trypsinized and analyzed by shotgun nLC-MS/MS. Proteins that were significantly different in abundance between SB623- and corresponding MSC-ECM are plotted in (A), with the exception of fibronectin (FN1), which is plotted in (B) because of its relatively high abundance. Collagen 1 alpha 1 (COL1A1), collagen 6 alpha 1 (COL6A1), collagen 6 alpha 3 (COL6A3), perlecan (HSPG2), latent transforming growth factor binding protein 1 (LTBP1), tenascin-c (TNC), transforming growth factor-beta-induced protein ig-h3 (TGFBI), transglutaminase 2 (TGM2). Mean ± SD; *p-value <0.05.
Figure 5.
Differences in gene expression levels between SB623 and MSC are consistent with differences detected using SAISD-nLC-MS/MS.
Gene expression levels were quantified using qRT-PCR and normalized to the expression of GAPDH; normalized values for MSC were set to 1 and values for SB623 were expressed relative to MSC. Four proteins were analyzed: perlecan (HSPG2), tenascin-C (TNC), transglutaminase 2 (TGM2), and latent-transforming growth factor beta-binding protein 1 (LTBP1). The qRT-PCR data was taken from 5 different human donor pairs, two of which were the same (donors 3 and 4) as in Figure 2, one SAISD-nLC-MS/MS donor (Table 2) and two unassociated donors. Mean ± SD.