Tandem mass tags

In the model and the DZXI groups, tissues (four replicates in each group) around the ischemic core were harvested after 7 days of treatments. The samples were digested with trypsin and labelled with TMT (tandem mass tags, Thermo). The same amount of each labelled sample was mixed, chromatographically separation, and finally subjected to an LC-MS/MS analysis. The peptide mass maps were analysed using Proteome Discoverer^TM2.2 software (Thermo) and the UniProt database. The significantly different proteins between the two groups were identified with fold change (FC) >1.5 and p value (calculated by t-test) < 0.05. These differentially expressed proteins were entered as foci into the subsequent clustering analysis, GO analysis and molecular networks generated from the STRING database.

The total protein from mouse aortas was isolated, and the concentration was measured using BCA for proteomics analysis. 50 μg of protein was taken from each sample and adjusted to the same concentration and volume. Dithiothreitol (DTT) was added to the above protein solution to make the final concentration of DTT 5 mM, and incubated at 55 °C for 30 min. Add the appropriate volume of iodoacetamide to make a final concentration of 10 mM and then 6 times the volume of acetone to the above solution to precipitate the protein at -20 °C overnight. The precipitate was collected by centrifugation and digested overnight at 37 °C with trypsin. Tryptic peptides were vacuum-dried and labeled using Tandem Mass Tags (Thermo, USA) under the manufacturer’s instructions. Peptide fragments were separated by high pH liquid chromatographic and then analyzed by MS. Data were analyzed using Proteome Discover 2.4.1.15 (Thermo, USA). The p-values of trusted proteins calculated with Student’s t-test showed significant differences (fold-change ≥1.2 and p-value < 0.05) between groups. The biological function of the proteins was analyzed using KEGG and Gene Ontology databases.

Purified LTBP4S-A (80 μg) was incubated for 25 h with 70 nm purified ADAMTS7-T8 in 50 mm HEPES, pH 7.5, 5 mm CaCl₂ in the presence or absence of broad-spectrum metalloprotease inhibitor GM6001 (90 μm). Samples were analyzed by SDS-PAGE/Coomassie Blue to confirm proteolysis and the absence thereof in the cleavage and control condition, respectively. New N termini generated by ADAMTS7 were labeled with Tandem Mass Tags (Thermo Fisher) according to the manufacturer's instructions prior to incomplete digest with trypsin (Thermo Fisher), chymotrypsin (Thermo Fisher) and Glu-C (Thermo Fisher). LC-MS/MS was performed at the Proteomic Facility of the University of Liège using an ACQUITY UPLC M-Class System (Waters) hyphenated to a Q Exactive (Thermo Scientific), in nanoelectrospray positive ion mode. Data were analyzed with Proteome Discoverer version. 2.1.1.21. The protein/peptide identifications were performed against a bovine background protein database supplemented with the sequence of the human target protein LTBP4. Search parameters were set as “no Enzyme” because of the specific multi-enzymatic limited digestion that was applied. Scissile bonds were derived from peptides labeled with TMT at the N terminus that were identified in the active protease condition, but not in the control condition. All reported peptides have a false discovery rate equal or lower than 0.01.