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Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

Prudova A, auf dem Keller U, Butler GS, Overall CM - Mol. Cell Proteomics (2010)

Bottom Line: We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins.Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2.Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Centre for Blood Research, University of British Columbia, 4.401 Life Sciences Institute, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.

ABSTRACT
Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

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Biochemical validation of MMP-2 and MMP-9 substrates. Recombinant proteins identified by iTRAQ-TAILS as high confidence or potential substrates were incubated for 18 h at 37 °C with MMP-2, MMP-9, or buffer alone, or MMP-2 and MMP-9 were incubated alone. A, digestion products of recombinant human Dkk-3 were separated by 10% SDS-PAGE. Cleavage fragments of recombinant human peptidyl-prolyl cis-trans isomerase A (PPI-A) (B) and CCL7 (C) were separated on 15% Tris-Tricine gels. Digestion products of recombinant pyruvate kinase M1/M2 (D) and human C1r subcomponent A (E) were separated by 10% SDS-PAGE. Digestion products were visualized by silver staining except for peptidyl-prolyl cis-trans isomerase A, which was detected by Western blotting using the corresponding rabbit polyclonal antibody. Arrows indicate major cleavage products. C1r protein was degraded by MMP-2 as shown by loss of the intact protein band. Autolysis of the MMP-2 and MMP-9 proteases during the 18-h assay often occurred and is typical, although the proteases used were purified to single band homogeneity as shown in supplemental Fig. 2. Pyr, pyruvate.
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Figure 5: Biochemical validation of MMP-2 and MMP-9 substrates. Recombinant proteins identified by iTRAQ-TAILS as high confidence or potential substrates were incubated for 18 h at 37 °C with MMP-2, MMP-9, or buffer alone, or MMP-2 and MMP-9 were incubated alone. A, digestion products of recombinant human Dkk-3 were separated by 10% SDS-PAGE. Cleavage fragments of recombinant human peptidyl-prolyl cis-trans isomerase A (PPI-A) (B) and CCL7 (C) were separated on 15% Tris-Tricine gels. Digestion products of recombinant pyruvate kinase M1/M2 (D) and human C1r subcomponent A (E) were separated by 10% SDS-PAGE. Digestion products were visualized by silver staining except for peptidyl-prolyl cis-trans isomerase A, which was detected by Western blotting using the corresponding rabbit polyclonal antibody. Arrows indicate major cleavage products. C1r protein was degraded by MMP-2 as shown by loss of the intact protein band. Autolysis of the MMP-2 and MMP-9 proteases during the 18-h assay often occurred and is typical, although the proteases used were purified to single band homogeneity as shown in supplemental Fig. 2. Pyr, pyruvate.

Mentions: Pyruvate kinase M1/M2 was identified as a high confidence MMP-2 substrate and as a candidate MMP-9 substrate as evident from three2 distinct peptides, 17LHAAMADTFLEHMCR (supplemental Table 12, peptide 148), 21MADTFLEHMCR (supplemental Table 12, peptide 141), and 109VALDTKGPEIR (supplemental Table 12, peptide 173), with MMP-2 iTRAQ ratios of 15.27, 16.08, and 13.11 and MMP-9 iTRAQ ratios of 2.15, 1.41, and 1.62, respectively. Incubation of pyruvate kinase M1/M2 with MMP-2 and MMP-9 resulted in loss of intensity of the full-length protein (more so with MMP-2 than with MMP-9) and appearance of multiple lower molecular weight fragments with distinct pattern differences between the two MMPs (Fig. 5).


Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

Prudova A, auf dem Keller U, Butler GS, Overall CM - Mol. Cell Proteomics (2010)

Biochemical validation of MMP-2 and MMP-9 substrates. Recombinant proteins identified by iTRAQ-TAILS as high confidence or potential substrates were incubated for 18 h at 37 °C with MMP-2, MMP-9, or buffer alone, or MMP-2 and MMP-9 were incubated alone. A, digestion products of recombinant human Dkk-3 were separated by 10% SDS-PAGE. Cleavage fragments of recombinant human peptidyl-prolyl cis-trans isomerase A (PPI-A) (B) and CCL7 (C) were separated on 15% Tris-Tricine gels. Digestion products of recombinant pyruvate kinase M1/M2 (D) and human C1r subcomponent A (E) were separated by 10% SDS-PAGE. Digestion products were visualized by silver staining except for peptidyl-prolyl cis-trans isomerase A, which was detected by Western blotting using the corresponding rabbit polyclonal antibody. Arrows indicate major cleavage products. C1r protein was degraded by MMP-2 as shown by loss of the intact protein band. Autolysis of the MMP-2 and MMP-9 proteases during the 18-h assay often occurred and is typical, although the proteases used were purified to single band homogeneity as shown in supplemental Fig. 2. Pyr, pyruvate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871422&req=5

Figure 5: Biochemical validation of MMP-2 and MMP-9 substrates. Recombinant proteins identified by iTRAQ-TAILS as high confidence or potential substrates were incubated for 18 h at 37 °C with MMP-2, MMP-9, or buffer alone, or MMP-2 and MMP-9 were incubated alone. A, digestion products of recombinant human Dkk-3 were separated by 10% SDS-PAGE. Cleavage fragments of recombinant human peptidyl-prolyl cis-trans isomerase A (PPI-A) (B) and CCL7 (C) were separated on 15% Tris-Tricine gels. Digestion products of recombinant pyruvate kinase M1/M2 (D) and human C1r subcomponent A (E) were separated by 10% SDS-PAGE. Digestion products were visualized by silver staining except for peptidyl-prolyl cis-trans isomerase A, which was detected by Western blotting using the corresponding rabbit polyclonal antibody. Arrows indicate major cleavage products. C1r protein was degraded by MMP-2 as shown by loss of the intact protein band. Autolysis of the MMP-2 and MMP-9 proteases during the 18-h assay often occurred and is typical, although the proteases used were purified to single band homogeneity as shown in supplemental Fig. 2. Pyr, pyruvate.
Mentions: Pyruvate kinase M1/M2 was identified as a high confidence MMP-2 substrate and as a candidate MMP-9 substrate as evident from three2 distinct peptides, 17LHAAMADTFLEHMCR (supplemental Table 12, peptide 148), 21MADTFLEHMCR (supplemental Table 12, peptide 141), and 109VALDTKGPEIR (supplemental Table 12, peptide 173), with MMP-2 iTRAQ ratios of 15.27, 16.08, and 13.11 and MMP-9 iTRAQ ratios of 2.15, 1.41, and 1.62, respectively. Incubation of pyruvate kinase M1/M2 with MMP-2 and MMP-9 resulted in loss of intensity of the full-length protein (more so with MMP-2 than with MMP-9) and appearance of multiple lower molecular weight fragments with distinct pattern differences between the two MMPs (Fig. 5).

Bottom Line: We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins.Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2.Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Centre for Blood Research, University of British Columbia, 4.401 Life Sciences Institute, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.

ABSTRACT
Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

Show MeSH
Related in: MedlinePlus