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

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.

Fig. 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).

Fig. 2.

iTRAQ-TAILS validation using simple chemokine mixture: examples of identified peptides and their iTRAQ ratios. A mixture of 13 chemokines (see supplemental Table 1 for the complete list) was divided in two and incubated with Glu-C or buffer alone. After TAILS and analysis by tandem mass spectrometry, the data were analyzed using Mascot and X! Tandem followed by validation with iProphet. The sequences of the chemokines CCL13 (A) and the first 52 residues of CXCL1, -2, and -3 (B) are shown with the peptides identified by TAILS and their corresponding Glu-C/control iTRAQ ratios indicated above. Peptides shown in red correspond to cleavage fragments generated by Glu-C processing after Asp, Glu, and deamidated Gln and Asn (indicated in bold). Peptides in blue represent original protein N-termini. * indicates pyro-Gln as identified by TAILS. The sequence ⁴⁰VIATLKNGR⁴⁸ in CXCL1 was identified as ⁴⁰VIATLKDGR⁴⁸, indicative of Asn⁴⁶ deamidation.

Fig. 3.

N-terminome analysis of Mmp2−/− fibroblast secretomes by iTRAQ-TAILS. Proteins secreted by Mmp2−/− murine fibroblasts were incubated with APMA-activated human MMP-2, MMP-9, 100 μm APMA, or buffer alone. The samples were processed by TAILS and analyzed by MS/MS. A, distribution of nonredundant peptides in the aliquot of the sample analyzed by mass spectrometry before (pre-TAILS; n = 1,197 peptides) and after N-terminal enrichment (TAILS; n = 3,152 peptides). In the pre-TAILS and TAILS analyses, 95 and 97% of all identified naturally acetylated peptides, respectively, were found to contain ≥1 lysine residue(s), which was iTRAQ-labeled and therefore quantifiable. B, distribution of N-terminal modifications among original N-termini identified by TAILS analysis (n = 190 proteins). C, frequency distributions of acetylated amino acids at the protein N-termini and of residues found at non-blocked protein N-termini after initiator methionine removal.

Fig. 4.

Galectin-1 and IGFBP-4 are novel substrates of MMP-9. A, summary of galectin-1 peptides and their corresponding iTRAQ ratios identified by iTRAQ-TAILS analysis in fibroblast secretomes treated with MMP-2 or MMP-9. B, the peptides identified by iTRAQ-TAILS are highlighted within the sequence of human galectin-1. Peptides with iTRAQ ratios ≥10 were identified as high confidence cleavage fragments and are shown in red. The peptide representing the mature original N-terminus is shown in blue. The neo-N-termini due to proteolysis by MMP-2 are indicated by red arrowheads, and those generated by proteolysis by other proteases in the sample, as suggested by the lower ratios, are indicated by black arrowheads. C, recombinant human galectin-1 was incubated for 18 h at 37 °C at a 10:1 molar ratio with MMP-2, MMP-9, or buffer alone, or MMP-2 and MMP-9 were incubated alone. The digestion products were separated on a 15% Tris-Tricine gel and visualized by silver staining. Arrows indicate cleavage fragments of galectin-1. D, summary of IGFBP-4 peptides identified by Edman sequencing in E and their corresponding iTRAQ ratios identified by iTRAQ-TAILS analysis of fibroblast secretomes treated with MMP-2 or MMP-9. E, recombinant human IGFBP-4 was incubated for 18 h at 37 °C at a 10:1 molar ratio with MMP-2, MMP-9, or buffer alone, or MMP-2 and MMP-9 were incubated alone as indicated. The digestion products were separated on a 15% Tris-Tricine gel followed by transfer to the PVDF membrane, R-250 Coomassie Blue staining, and Edman sequencing of the visible cleavage fragments. Cleavage products with their corresponding sequences identified by Edman degradation are indicated. ctr, control.

Fig. 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.

Fig. 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.

Fig. 7.

MMP-2 and MMP-9 substrate specificity. Heat maps (left panels) and protein sequence logos (right panels) for amino acid occurrences in P4–P4′ for neo-N-termini generated by MMP-2 (A) and MMP-9 (B) proteolysis (n = 201 for MMP-2 and n = 19 for MMP-9) are shown. Protein sequence logos were generated using the iceLogo software package with correction for natural amino acid abundance (44).