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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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Related in: MedlinePlus

iTRAQ-TAILS workflow. Multiple samples (two up to eight) can be co-analyzed and compared using iTRAQ-TAILS. In the schematic, a proteolyzed protein is indicated by a black star in samples 2, 3, and 4 generated by different proteases (or under different conditions). The proteins in each complex sample are labeled with distinct iTRAQ labels (that give 114, 115, 116, or 117 reporter ions in MS2 mode) using whole protein iTRAQ labeling on their free N-termini and lysine residues (represented by colored peptides). This not only distinguishes proteins derived from each sample (indicated by different colors) but can also be used for conventional iTRAQ quantification of the proteins present after trypsinization. Pooled samples are subjected to trypsin digestion to generate the following peptides: N-terminal peptides that are modified by iTRAQ reagents or pre-existing modifications such as acetylation (Ac) (indicated by color) or internal and C-terminal peptides having free N-termini (indicated in gray). The peptides are pooled, and the N-terminal peptides from the original proteins are isolated by negative selection: a polyaldehyde dendritic polymer binds all of the peptides with free (trypsin-generated) N-termini but not the peptides with blocked N-termini (iTRAQ-labeled or naturally acetylated or cyclized). The polymer and bound peptides are removed by ultrafiltration, and the N-terminal peptides are subjected to MS/MS. In MS2, following peptide fragmentation, the iTRAQ labels are quantified, giving the relative amount of peptide derived from each sample, whereas the rest of the b and y ions permit peptide identification using the search engines Mascot and X! Tandem. Original N-termini present in all samples give an iTRAQ ratio centered on 1.0. The appearance of a singleton reporter ion represents the neo-N-terminus (indicated by the black star), which has an iTRAQ ratio significantly >1. Alternatively, indirect evidence for proteolysis is revealed by a disappearance of the original N-terminus (which will have an iTRAQ ratio <1).
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Figure 1: iTRAQ-TAILS workflow. Multiple samples (two up to eight) can be co-analyzed and compared using iTRAQ-TAILS. In the schematic, a proteolyzed protein is indicated by a black star in samples 2, 3, and 4 generated by different proteases (or under different conditions). The proteins in each complex sample are labeled with distinct iTRAQ labels (that give 114, 115, 116, or 117 reporter ions in MS2 mode) using whole protein iTRAQ labeling on their free N-termini and lysine residues (represented by colored peptides). This not only distinguishes proteins derived from each sample (indicated by different colors) but can also be used for conventional iTRAQ quantification of the proteins present after trypsinization. Pooled samples are subjected to trypsin digestion to generate the following peptides: N-terminal peptides that are modified by iTRAQ reagents or pre-existing modifications such as acetylation (Ac) (indicated by color) or internal and C-terminal peptides having free N-termini (indicated in gray). The peptides are pooled, and the N-terminal peptides from the original proteins are isolated by negative selection: a polyaldehyde dendritic polymer binds all of the peptides with free (trypsin-generated) N-termini but not the peptides with blocked N-termini (iTRAQ-labeled or naturally acetylated or cyclized). The polymer and bound peptides are removed by ultrafiltration, and the N-terminal peptides are subjected to MS/MS. In MS2, following peptide fragmentation, the iTRAQ labels are quantified, giving the relative amount of peptide derived from each sample, whereas the rest of the b and y ions permit peptide identification using the search engines Mascot and X! Tandem. Original N-termini present in all samples give an iTRAQ ratio centered on 1.0. The appearance of a singleton reporter ion represents the neo-N-terminus (indicated by the black star), which has an iTRAQ ratio significantly >1. Alternatively, indirect evidence for proteolysis is revealed by a disappearance of the original N-terminus (which will have an iTRAQ ratio <1).

Mentions: Here we modified our recently published TAILS protocol (32) with the incorporation of iTRAQ labeling for the multiplexing of four to eight samples per analysis that allows for more accurate quantification and versatile experimental designs (Fig. 1). Unlike conventional iTRAQ where labeling is performed after trypsin digestion on peptides (45), we aimed to quantify cleavage events in native proteins and so label and quantify these events at the protein level rather than at the peptide level. To do so, we devised a novel protocol for efficient, rapid, and amino group-specific protein iTRAQ labeling performed before trypsinization. This represents a key step of the new TAILS workflow as it (i) blocks protein N-termini, allowing for their subsequent enrichment by negative selection along with all naturally blocked N-termini, such as those with acetylated or cyclized N-termini (32); (ii) simultaneously introduces a stable isotope tag to α-amino groups of protein N-termini and ε-amino groups of lysine residues, thus allowing for accurate quantification; and (iii) because trypsin cannot cleave at iTRAQ-blocked lysine residues, trypsinization yields peptides with Arg-C-like specificity, lengthening most protease-truncated semitryptic neo-N-peptides for improved MS/MS identification and substrate coverage. Our highly optimized conditions, using 50% DMSO as a solvent, yielded 97 ± 3% labeling efficiency within 30 min for protein N-terminal α-amines and lysine ε-amines with minimal side reactions on Tyr, Ser, or Thr (when the data sets were searched using iTRAQ-Tyr, -Ser, or -Thr as variable modifications). Hence, this whole protein iTRAQ labeling method has applicability for other applications aimed at protein characterization.


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)

iTRAQ-TAILS workflow. Multiple samples (two up to eight) can be co-analyzed and compared using iTRAQ-TAILS. In the schematic, a proteolyzed protein is indicated by a black star in samples 2, 3, and 4 generated by different proteases (or under different conditions). The proteins in each complex sample are labeled with distinct iTRAQ labels (that give 114, 115, 116, or 117 reporter ions in MS2 mode) using whole protein iTRAQ labeling on their free N-termini and lysine residues (represented by colored peptides). This not only distinguishes proteins derived from each sample (indicated by different colors) but can also be used for conventional iTRAQ quantification of the proteins present after trypsinization. Pooled samples are subjected to trypsin digestion to generate the following peptides: N-terminal peptides that are modified by iTRAQ reagents or pre-existing modifications such as acetylation (Ac) (indicated by color) or internal and C-terminal peptides having free N-termini (indicated in gray). The peptides are pooled, and the N-terminal peptides from the original proteins are isolated by negative selection: a polyaldehyde dendritic polymer binds all of the peptides with free (trypsin-generated) N-termini but not the peptides with blocked N-termini (iTRAQ-labeled or naturally acetylated or cyclized). The polymer and bound peptides are removed by ultrafiltration, and the N-terminal peptides are subjected to MS/MS. In MS2, following peptide fragmentation, the iTRAQ labels are quantified, giving the relative amount of peptide derived from each sample, whereas the rest of the b and y ions permit peptide identification using the search engines Mascot and X! Tandem. Original N-termini present in all samples give an iTRAQ ratio centered on 1.0. The appearance of a singleton reporter ion represents the neo-N-terminus (indicated by the black star), which has an iTRAQ ratio significantly >1. Alternatively, indirect evidence for proteolysis is revealed by a disappearance of the original N-terminus (which will have an iTRAQ ratio <1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: iTRAQ-TAILS workflow. Multiple samples (two up to eight) can be co-analyzed and compared using iTRAQ-TAILS. In the schematic, a proteolyzed protein is indicated by a black star in samples 2, 3, and 4 generated by different proteases (or under different conditions). The proteins in each complex sample are labeled with distinct iTRAQ labels (that give 114, 115, 116, or 117 reporter ions in MS2 mode) using whole protein iTRAQ labeling on their free N-termini and lysine residues (represented by colored peptides). This not only distinguishes proteins derived from each sample (indicated by different colors) but can also be used for conventional iTRAQ quantification of the proteins present after trypsinization. Pooled samples are subjected to trypsin digestion to generate the following peptides: N-terminal peptides that are modified by iTRAQ reagents or pre-existing modifications such as acetylation (Ac) (indicated by color) or internal and C-terminal peptides having free N-termini (indicated in gray). The peptides are pooled, and the N-terminal peptides from the original proteins are isolated by negative selection: a polyaldehyde dendritic polymer binds all of the peptides with free (trypsin-generated) N-termini but not the peptides with blocked N-termini (iTRAQ-labeled or naturally acetylated or cyclized). The polymer and bound peptides are removed by ultrafiltration, and the N-terminal peptides are subjected to MS/MS. In MS2, following peptide fragmentation, the iTRAQ labels are quantified, giving the relative amount of peptide derived from each sample, whereas the rest of the b and y ions permit peptide identification using the search engines Mascot and X! Tandem. Original N-termini present in all samples give an iTRAQ ratio centered on 1.0. The appearance of a singleton reporter ion represents the neo-N-terminus (indicated by the black star), which has an iTRAQ ratio significantly >1. Alternatively, indirect evidence for proteolysis is revealed by a disappearance of the original N-terminus (which will have an iTRAQ ratio <1).
Mentions: Here we modified our recently published TAILS protocol (32) with the incorporation of iTRAQ labeling for the multiplexing of four to eight samples per analysis that allows for more accurate quantification and versatile experimental designs (Fig. 1). Unlike conventional iTRAQ where labeling is performed after trypsin digestion on peptides (45), we aimed to quantify cleavage events in native proteins and so label and quantify these events at the protein level rather than at the peptide level. To do so, we devised a novel protocol for efficient, rapid, and amino group-specific protein iTRAQ labeling performed before trypsinization. This represents a key step of the new TAILS workflow as it (i) blocks protein N-termini, allowing for their subsequent enrichment by negative selection along with all naturally blocked N-termini, such as those with acetylated or cyclized N-termini (32); (ii) simultaneously introduces a stable isotope tag to α-amino groups of protein N-termini and ε-amino groups of lysine residues, thus allowing for accurate quantification; and (iii) because trypsin cannot cleave at iTRAQ-blocked lysine residues, trypsinization yields peptides with Arg-C-like specificity, lengthening most protease-truncated semitryptic neo-N-peptides for improved MS/MS identification and substrate coverage. Our highly optimized conditions, using 50% DMSO as a solvent, yielded 97 ± 3% labeling efficiency within 30 min for protein N-terminal α-amines and lysine ε-amines with minimal side reactions on Tyr, Ser, or Thr (when the data sets were searched using iTRAQ-Tyr, -Ser, or -Thr as variable modifications). Hence, this whole protein iTRAQ labeling method has applicability for other applications aimed at protein characterization.

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