PMC

Protein categories. The classification and numerical distribution of the identified extracellular proteins in the NaCl (A), guanidine (B), and SDS (C) extracts are shown. The size of the doughnut charts is representative of the number of extracellular space proteins identified in the different extracts.

Evaluation of RePlay analysis. Representative chromatograms for a NaCl (A) and a guanidine extract (B) using RePlay are shown. The RePlay device allows the reanalysis of the same sample during a single LC-MS/MS run. The combined correlation of the spectral counts obtained by RePlay in the three NaCl extracts (C) and in the three guanidine extracts (D) demonstrates the good correlation between the technical replicates (Correlation for each of the six extrats can be found in supplemental Fig. 3.). No RePlay analysis was performed for the SDS extracts.

Confirmation of podocan, sclerostin, and agrin in aortic ECM. Immunohistological staining confirmed the presence of podocan in the aortic intima (A). Immunoreactivity appears in brown (magnification, 20×; inset magnification, 60×). Nuclei are counterstained with Mayer's hematoxylin (blue). Sclerostin (C) was localized in the subendothelial layer of the aortic intima. Finally, immunohistochemical staining confirmed the presence of agrin (E). Agrin is localized on the aortic endothelium. The respective isotype negative controls are shown in B, D, and F, respectively. The specificity of the sclerostin and agrin antibodies was confirmed by immunoblotting (data not shown). The aortic specimens were from a 44-year old female (A, B) and a 55-year old male patient (C–F).

Validation of proteomics data. Gelatin zymography confirmed the presence of MMP-2 in one of the three NaCl extracts (A). Periostin (B, upper panel) and asporin (B, bottom panel) were validated by immunoblotting in the guanidine extracts (on the same blotting membrane; first, asporin; and second, reprobing for periostin). The total protein loading control for the extracts can be seen in C, bottom panel. For periostin and asporin, the relative band intensity of the immunoblots was measured, and the values were correlated with the spectral counts of the original and the RePlay analysis (n = 2, mean ± S.D. Error bars denote standard deviation.) (C and D).

Detection of proteolysis. Percent sequence coverage for fibronectin in the NaCl (A) and guanidine extracts (B) is shown. “R” denotes RePlay analysis. Note the increase of fibronectin products in the NaCl extracts of the fourth aortic specimen. Western immunoblotting of fibronectin in the NaCl (C) and guanidine (D) extracts confirmed the laddering of fibronectin. The product ion spectrum of the doubly charged tryptic peptide SLGPALLLLQK was identified as MMP-9 (E) (peptide probability, 95%; SEQUEST XCorr score, 2.67; SEQUEST ΔCn score, 0.40; observed m/z, 576.87; actual peptide mass, 1,151.73 Da; peptide charge, 2; delta amu, 0.0049; delta ppm, 4.3; peptide start index, 66; peptide stop index, 76). MMP-9 was only detected by LC-MS/MS in the NaCl extract of the fourth aortic specimen. Gelatin zymography confirmed higher levels of MMP-9 in this sample (C, bottom panel).

Identification of extracellular proteins in human aortas. A, schematic summary. Biochemical subfractionation was used for the enrichment of extracellular space proteins before proteomic analysis. B, validation of decellularization by hematoxylin/eosin staining. Representative images of a hematoxylin/eosin-stained human aorta before (left panel) and after decellularization with 0.08% SDS (right panel) are shown. L, lumen; I, intima; M, media. Arrows depict the intima/media border. Note the loss of nuclear (blue; hematoxylin) and cytoplasmic contents (pink; eosin), whereas other eosinophilic structures, such as collagen fibers, are preserved. C, immunoblotting and silver staining. The ECM glycoprotein AEBP1 is found in the NaCl and guanidine (Guan) extracts, which are enriched in proteins of the extracellular space (upper panel). Cytoplasmic β-actin is predominantly found in the SDS extracts (middle panel), demonstrating successful depletion of cellular contents. Silver staining of proteins from all extracts separated by one-dimensional SDS-PAGE shows the pattern of proteins extracted in the three different extraction steps (bottom panel).