Table II

Temporal changes in abundance levels of known and candidate MMP-2 substrates identified by iTRAQ proteomics in conditioned medium from active MMP-2-transfected cells

Shown are relative abundance ratios of iTRAQ-labeled tryptic peptides identified in the conditioned medium from Mmp2−/− fibroblasts transfected with ΔppMMP-2 (MMP-2) or ΔppE375AMMP-2 (E/A). Conditioned medium proteins were collected at 3 and 24 h (experiment 1), 24 h (experiment 2), or 3 and 48 h (experiment 3), and the mean of the MMP-2:E/A ratios for each iTRAQ-labeled peptide at these time points was calculated. A ratio of 1.0 indicates no change in abundance of protein detected in the MMP-2 and E/A samples; ratios >1.0 indicate relative accumulation of protein in conditioned medium; ratios <1.0 indicate relative depletion of protein from conditioned medium and were reflected by the smaller numbers of peptides identified per protein due to MMP-2 activity. A confidence level ≥99% was the inclusion criterion for identification of tryptic peptides. The number of unique iTRAQ-labeled tryptic peptides is shown in parentheses with different forms of the same peptide being counted as one peptide; multiple identifications of the same peptide were also counted as a single identity. Peptides having an iTRAQ ratio >30 were considered singletons as one peptide of the pair could not be detected above the background noise resulting in an inability to accurately determine the area of such a very low ion peak. This ratio depicts a large unquantifiable change and indicates a very strong substrate candidate. Full data sets from which these selected proteins were extracted are presented in Supplemental Tables S3, A, B, and C.—, no peptides detected.

Ref.MMP-2:E/AaMMP-2:E/Ab24 hMMP-2:E/Ac
3 h24 h3 h48 h
Known substratesd
    MCP-3 10 6.327.5 (1)2.08.3 (1)
    Fibronectin 46 3.621.2 (14)5.1 (16)3.69.0 (23)
    Collagen α1 (I) 47 3.2>30 (14)12.2 (7)2.312.9 (19)
    Collagen α2 (I) 47 3.6>30 (18)14.0 (11)3.214.7 (28)
    Collagen α1 (III) 48 2.918.1 (8)4.0 (6)2.411.6 (10)
    Collagen α1 (IV) 49 1.35.3 (1)2.310.8 (4)
    Collagen α2 (IV) 49 1.17.3 (3)
    Collagen α2 (V) 49 1.24.9 (2)1.7 (1)2.68.6 (8)
    Collagen α1 (VI) 16 3.423.1 (2)1.66.1 (7)
    Collagen α2 (VI)e 16 5.6 (1)1.215.9 (2)
    Collagen α3 (VI) 50 4.414.6 (3)2.010.7 (1)
    Decorin 51 9.1>30 (2)8.2 (5)9.111.9 (3)
    Galectin-3 34 4.45.9 (2)
    Dystroglycanf 52 1.10.5 (1)1.12.2 (4)
    Osteonectinf 53 2.76.0 (5)0.4 (5)0.90.5 (6)
    Amyloid β A4 54 0.50.5 (4)1.0 (4)0.80.8 (5)
Candidate substratesd
    Procollagen C-proteinase enhancerf>30>30 (6)>30 (8)4.78.8 (8)
    Osteopontin>30>30 (2)15.6 (4)5.1>30 (3)
    Periostin7.0>30 (1)4.6 (1)3.320.2 (7)
    Galectin-110.011.5 (3)2.7 (3)>30>30 (4)
    Biglycan4.4>30 (3)3.3 (3)2.63.9 (2)
    Hepatoma-derived growth factorf1.41.6 (1)1.1 (1)11.5>30 (2)
    Fractalkinef4.9>30 (4)
    Macrophage migration-inhibitory factor25.75.5 (1)9.911.0 (2)
    Extracellular matrix protein-12.118.9 (1)11.6 (2)1.816.8 (7)
    Mimecan8.8>30 (2)1.32.2 (1)
    Semaphorin-7A5.0 (3)2.512.2 (5)
    Quiescin Q60.74.8 (1)2.49.7 (4)
    Prosaposin1.88.2 (5)
    Perlecanf1.8>30 (2)10.2 (3)1.29.7 (4)
    Heat shock protein-90α>30>30 (2)4.3 (3)1.10.7 (2)
    (Heat shock protein-90βg)1.21.2 (8)1.2 (7)1.30.9 (11)
    Dermatopontine1.94.4 (2)
    Calgizzarin8.7>30 (2)1.51.5 (2)
    IGFBP-4e0.2 (1)0.90.3 (2)
    Thrombospondin-10.20.3 (1)0.5 (1)0.40.7 (2)
    Chondroitin sulfate proteoglycan NG20.30.4 (2)0.40.4 (3)
    Clusterin0.30.1 (3)0.3 (4)0.30.2 (6)
    Dickkopf-related protein-30.10.1 (1)0.02 (1)0.30.1 (3)
    NCAM-1h0.10.01 (1)0.1 (1)
  • a Data collected from experiment 1 using four iTRAQ labels.

  • b Data collected from experiment 2 using two iTRAQ labels.

  • c Data collected from experiment 3 using four iTRAQ labels.

  • d Abbreviations used in protein list: MCP, monocyte chemotactic protein; IGFBP-4, insulin-like growth factor-binding protein-4; NCAM-1, neural cell adhesion molecule-1.

  • e Proteins identified with one unique tryptic peptide at 95% confidence level and one at 99%.

  • f Proteins having highly dispersed peptide ratios and with one or more peptide with a ratio ≥4.0 were subjected to peptide mapping and are presented in Table III.

  • g Heat shock protein-90β is included to highlight the specific response of MMP-2 compared with heat shock protein-90α.

  • h NCAM-1 was designated as a candidate substrate because other family members were known substrates despite being identified by only one peptide.