Limits...
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.

Show MeSH

Related in: MedlinePlus

Thrombospondin-2 is a novel MMP-2 and MMP-9 substrate. A, recombinant human thrombospondin-2 (TSP2) was incubated for 18 h at 37 °C at a 10:1 molar ratio with MMP-2, MMP-9, or buffer alone. The digestion products were separated on by 10% SDS-PAGE and visualized by silver staining (left panel) or detected by Western blotting using an antibody raised against the N-terminal (middle panel) or C-terminal domain of the protein (right panel). B, fragments of thrombospondin-2 after incubation with MMP-2 were transferred to a PVDF membrane, and N-terminal sequences were derived by Edman degradation as indicated by arrows. ns indicates that no sequence was obtained. All lanes were from the same gel. C, summary of thrombospondin-2 peptides and corresponding iTRAQ ratios identified by iTRAQ-TAILS in fibroblast secretomes treated with MMP-2 or MMP-9. D, schematic diagram of thrombospondin-2. N-terminal, von Willebrand factor (VWF), thrombospondin (TSP) type I, epidermal growth factor (EGF), thrombospondin (TSP) type III, and C-terminal domains are shown. Positions of cleavage sites or the mature N-terminus of the murine protein identified by iTRAQ-TAILS are shown above, and those identified by Edman degradation of the human protein after digestion by human MMP-2 are shown below the protein. ctr, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2871422&req=5

Figure 6: Thrombospondin-2 is a novel MMP-2 and MMP-9 substrate. A, recombinant human thrombospondin-2 (TSP2) was incubated for 18 h at 37 °C at a 10:1 molar ratio with MMP-2, MMP-9, or buffer alone. The digestion products were separated on by 10% SDS-PAGE and visualized by silver staining (left panel) or detected by Western blotting using an antibody raised against the N-terminal (middle panel) or C-terminal domain of the protein (right panel). B, fragments of thrombospondin-2 after incubation with MMP-2 were transferred to a PVDF membrane, and N-terminal sequences were derived by Edman degradation as indicated by arrows. ns indicates that no sequence was obtained. All lanes were from the same gel. C, summary of thrombospondin-2 peptides and corresponding iTRAQ ratios identified by iTRAQ-TAILS in fibroblast secretomes treated with MMP-2 or MMP-9. D, schematic diagram of thrombospondin-2. N-terminal, von Willebrand factor (VWF), thrombospondin (TSP) type I, epidermal growth factor (EGF), thrombospondin (TSP) type III, and C-terminal domains are shown. Positions of cleavage sites or the mature N-terminus of the murine protein identified by iTRAQ-TAILS are shown above, and those identified by Edman degradation of the human protein after digestion by human MMP-2 are shown below the protein. ctr, control.

Mentions: Complement C1q is cleaved by MMP-1, -2, -3, and -9 (54) as is the related protein mannose binding lectin (55). We identified complement C1r-A subcomponent as a high confidence MMP-2 substrate with an iTRAQ ratio of ≥35 for the cleavage site VSS↓191LEY (supplemental Table 12, peptide 13). For MMP-9, C1r-A was only a candidate substrate because it had an iTRAQ ratio of 4.36, considerably less than the statistically defined cutoff ratio of 10 needed for high confidence substrate identification. Indeed, full-length C1r-A was degraded by MMP-2, which caused disappearance of the protein, but MMP-9 incubation had no effect on C1r-A (Fig. 5). This illustrates the higher false positive rate at lower iTRAQ ratios and underscores the importance of stringent statistically derived cutoffs for high confidence substrate identification rather than using arbitrary values such as 2-fold. Nonetheless, other lower iTRAQ ratio candidates that were successfully validated as novel MMP-9 substrates included dickkopf-related protein-3 (Fig. 5), previously shown to be processed by MMP-2 at an unknown site (19) but identified here, whereas the related dickkopf-1 protein was processed by MMP-14 (18). We also biochemically verified a known MMP-2 cleavage of peptidyl-prolyl cis-trans isomerase A (32) (Fig. 5) and confirmed a novel MMP-9 cleavage prompted by previous studies that detected processing of peptidyl-prolyl cis-trans isomerase A by MMP-14 (18). Human chemokine CC7 was used as a positive control for MMP-2 (Fig. 5), and we found for the first time that it too was cleaved by MMP-9. Finally, thrombospondin-1 and thrombospondin-2 are MMP-2 (19) and MMP-14 (18) substrates, respectively. Several neo-N-termini in thromspondin-2 were identified (supplemental Table 12), and its cleavage by MMP-2 and MMP-9 was confirmed (Fig. 6 and supplemental Fig. 4).


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)

Thrombospondin-2 is a novel MMP-2 and MMP-9 substrate. A, recombinant human thrombospondin-2 (TSP2) was incubated for 18 h at 37 °C at a 10:1 molar ratio with MMP-2, MMP-9, or buffer alone. The digestion products were separated on by 10% SDS-PAGE and visualized by silver staining (left panel) or detected by Western blotting using an antibody raised against the N-terminal (middle panel) or C-terminal domain of the protein (right panel). B, fragments of thrombospondin-2 after incubation with MMP-2 were transferred to a PVDF membrane, and N-terminal sequences were derived by Edman degradation as indicated by arrows. ns indicates that no sequence was obtained. All lanes were from the same gel. C, summary of thrombospondin-2 peptides and corresponding iTRAQ ratios identified by iTRAQ-TAILS in fibroblast secretomes treated with MMP-2 or MMP-9. D, schematic diagram of thrombospondin-2. N-terminal, von Willebrand factor (VWF), thrombospondin (TSP) type I, epidermal growth factor (EGF), thrombospondin (TSP) type III, and C-terminal domains are shown. Positions of cleavage sites or the mature N-terminus of the murine protein identified by iTRAQ-TAILS are shown above, and those identified by Edman degradation of the human protein after digestion by human MMP-2 are shown below the protein. ctr, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Thrombospondin-2 is a novel MMP-2 and MMP-9 substrate. A, recombinant human thrombospondin-2 (TSP2) was incubated for 18 h at 37 °C at a 10:1 molar ratio with MMP-2, MMP-9, or buffer alone. The digestion products were separated on by 10% SDS-PAGE and visualized by silver staining (left panel) or detected by Western blotting using an antibody raised against the N-terminal (middle panel) or C-terminal domain of the protein (right panel). B, fragments of thrombospondin-2 after incubation with MMP-2 were transferred to a PVDF membrane, and N-terminal sequences were derived by Edman degradation as indicated by arrows. ns indicates that no sequence was obtained. All lanes were from the same gel. C, summary of thrombospondin-2 peptides and corresponding iTRAQ ratios identified by iTRAQ-TAILS in fibroblast secretomes treated with MMP-2 or MMP-9. D, schematic diagram of thrombospondin-2. N-terminal, von Willebrand factor (VWF), thrombospondin (TSP) type I, epidermal growth factor (EGF), thrombospondin (TSP) type III, and C-terminal domains are shown. Positions of cleavage sites or the mature N-terminus of the murine protein identified by iTRAQ-TAILS are shown above, and those identified by Edman degradation of the human protein after digestion by human MMP-2 are shown below the protein. ctr, control.
Mentions: Complement C1q is cleaved by MMP-1, -2, -3, and -9 (54) as is the related protein mannose binding lectin (55). We identified complement C1r-A subcomponent as a high confidence MMP-2 substrate with an iTRAQ ratio of ≥35 for the cleavage site VSS↓191LEY (supplemental Table 12, peptide 13). For MMP-9, C1r-A was only a candidate substrate because it had an iTRAQ ratio of 4.36, considerably less than the statistically defined cutoff ratio of 10 needed for high confidence substrate identification. Indeed, full-length C1r-A was degraded by MMP-2, which caused disappearance of the protein, but MMP-9 incubation had no effect on C1r-A (Fig. 5). This illustrates the higher false positive rate at lower iTRAQ ratios and underscores the importance of stringent statistically derived cutoffs for high confidence substrate identification rather than using arbitrary values such as 2-fold. Nonetheless, other lower iTRAQ ratio candidates that were successfully validated as novel MMP-9 substrates included dickkopf-related protein-3 (Fig. 5), previously shown to be processed by MMP-2 at an unknown site (19) but identified here, whereas the related dickkopf-1 protein was processed by MMP-14 (18). We also biochemically verified a known MMP-2 cleavage of peptidyl-prolyl cis-trans isomerase A (32) (Fig. 5) and confirmed a novel MMP-9 cleavage prompted by previous studies that detected processing of peptidyl-prolyl cis-trans isomerase A by MMP-14 (18). Human chemokine CC7 was used as a positive control for MMP-2 (Fig. 5), and we found for the first time that it too was cleaved by MMP-9. Finally, thrombospondin-1 and thrombospondin-2 are MMP-2 (19) and MMP-14 (18) substrates, respectively. Several neo-N-termini in thromspondin-2 were identified (supplemental Table 12), and its cleavage by MMP-2 and MMP-9 was confirmed (Fig. 6 and supplemental Fig. 4).

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