Fig 2: Distinct sequence motifs enriched/de-enriched in ventricular extracts with MMP-2-Fc-induced proteolysis. Sequence motifs of the identified cleavage sites are shown for all identified cleavage sites as IceLogo’s with a p-value threshold of <0.05. The IceLogos depict the enriched sequence surrounding the proteolytic cleavage site between P1 and P1′ (P1↓P1′).39 Enriched and de-enriched amino acid consensus sequences are shown for (A) all cleavage sites and (B) unique cleavage sites, identified in ventricular extracts from control hearts without added protease. Enriched and de-enriched amino acid consensus sequences are shown for (C) all cleavage sites and (D) unique cleavage sites, identified in ventricular extracts incubated with MMP-2-Fc. Consensus sequences were generated using IceLogo.

Fig 3: Engineering of a recombinant MMP-2 Fc-fusion protein that is enzymatically active. Catalytically active recombinant MMP-2-Fc is expressed in mammalian cells. (A) MMP-2 contains a pro-domain (pink) which must be removed or chemically altered for proteolytic activity. (B) Activity of MMP-2-Fc is monitored using fluorescent gelatin conjugate as a substrate. MMP-2-Fc is active and is inhibited by ARP-100, an MMP-2 selective inhibitor, in a concentration-dependent manner. The graph depicted is an average of three replicates, n = 3. (C) Activity of MMP-2-Fc in ventricular extracts is monitored using a fluorescent gelatin conjugate as a substrate and compared to the control (no MMP-2) and a positive control of MMP-2-Fc in activity buffer.

Fig 4: Proteolysis of MDHM and PGK1 by MMP-2-Fc in vitro. To further characterize the substrates identified using N-terminomics, the proteolysis of mitochondrial malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1) was monitored. (A) The location of the cleavage sites identified by N-terminomics highlighted on the structure of MDHM (PDB 2DFD). (B) Ventricular extracts incubated with MMP-2-Fc for 0–18 h. A negative control containing the MMP-2 inhibitor APR-100 is included. The disappearance of full-length (FL) MDHM at ∼36 kDa is time-dependent. Total protein staining was used as a loading control. (C) Quantification of the relative abundance of full-length MDHM is reported as mean ± SEM. (D) The location of the cleavage sites identified by N-terminomics highlighted on the structure of PGK1 (PDB ID 4O3F). (E) Recombinant mouse PGK1 was incubated with MMP-2-Fc for 0–18 h. A negative control containing EDTA to inhibit MMP-2-Fc was included. The disappearance of full-length (FL) PGK1 (46 kDa) dependent on MMP-2-Fc incubation was visualized by gel electrophoresis and Coomassie staining. No cleavage products were detected. (F) Quantification of the abundance of full-length (FL) PGK1 is reported as mean ± SEM. These results are representative of 3 independent experiments. ***p < 0.001, *p < 0.05, #p < 0.06, by unpaired Student’s t test.

Fig 5: Proteolytic changes in ventricular extracts following treatment with MMP-2-Fc. N-terminomic labeling enables the identification of proteolytic sites following incubation with recombinant MMP-2-Fc. (A) Venn diagram depicting the overlap between control ventricular extracts (without MMP-2-Fc) and those with MMP-2-Fc (the reaction was repeated three times for control and five times for MMP-2-Fc. Positive identifications were cleavage sites identified in at least two of the replicates). A total of 132 unique cleavage sites were identified in at least two replicates. 95 cleavage sites were only identified in extracts incubated with MMP-2-Fc, 2 cleavage sites were only identified in control extracts, and 35 were identified in both control extracts and those incubated with MMP-2-Fc. (B) Some substrates were cleaved at more than one site. The 95 cleavage sites identified in MMP-2-Fc extracts correspond to 68 substrates. Twenty-nine substrates were identified in both sample groups. (C) Enriched gene ontology pathways in each group are depicted as a bar graph. Distinct gene ontology pathways are enriched in cleavage sites identified following proteolysis by MMP-2-Fc (blue) compared to both (gray) with a significant p-value < 0.05. Gene ontology was performed on unique proteins from each group. A detailed list of proteins and gene ontology analysis is provided in Supporting Data 2.