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A statistics-based platform for quantitative N-terminome analysis and identification of protease cleavage products.

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

Bottom Line: Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode.The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF).By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed.

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
Terminal amine isotopic labeling of substrates (TAILS), our recently introduced platform for quantitative N-terminome analysis, enables wide dynamic range identification of original mature protein N-termini and protease cleavage products. Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode. The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF). Furthermore, the isoform assignment score (IAS) is introduced, a new scoring system for the evaluation of single peptide-to-protein assignments based on high confidence protein identifications in the same sample prior to negative selection enrichment of N-terminal peptides. By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed.

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Processing of murine proEMAP/p43 by MMP-2. A, amino acid sequence of proEMAP/p43 showing the acetylated natural N-terminus of the mature protein (green) and an MMP-2-generated neo-N-terminus (blue) identified by CLIP-TRAQ-TAILS. B, mass spectra assigned to the natural N-terminus and neo-N-terminus of proEMAP/p43, respectively. Insets show a close-up of the CLIP-TRAQ reporter ion region indicating the presence of the natural N-terminal peptide in both samples and the neo-N-terminus only in the MMP-2-treated (CLIP-TRAQ-113) sample. C, MMP-2 cleavage of proEMAP/p43 analyzed by 15% Tris-Tricine SDS-PAGE. Arrows show full-length and cleavage products of proEMAP/p43.
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Figure 5: Processing of murine proEMAP/p43 by MMP-2. A, amino acid sequence of proEMAP/p43 showing the acetylated natural N-terminus of the mature protein (green) and an MMP-2-generated neo-N-terminus (blue) identified by CLIP-TRAQ-TAILS. B, mass spectra assigned to the natural N-terminus and neo-N-terminus of proEMAP/p43, respectively. Insets show a close-up of the CLIP-TRAQ reporter ion region indicating the presence of the natural N-terminal peptide in both samples and the neo-N-terminus only in the MMP-2-treated (CLIP-TRAQ-113) sample. C, MMP-2 cleavage of proEMAP/p43 analyzed by 15% Tris-Tricine SDS-PAGE. Arrows show full-length and cleavage products of proEMAP/p43.

Mentions: As a second example, proEMAP/p43, a member of the small inducible cytokine family, was identified by an acetylated N-terminus after removal of the initiator methionine (Fig. 5, A and B). Although the corresponding Swiss-Prot entry (P31230) lacks this information, it was also predicted by the TermiNator algorithm (58) with 83% confidence. Importantly, the TAILS negative selection strategy allows the identification of acetylated N-termini that are proposed for up to 70% of all proteins (59) that positive selection strategies cannot detect. In addition, isotopic labeling of lysine side chains preserves quantification data provided the naturally blocked N-terminus harbors a lysine in its sequence as demonstrated for this example. Because CLIP-TRAQ labeling is at the protein level with trypsinization performed afterward, cleavage only occurs at arginine residues, preserving the label and increasing the size of the peptide and thus the probability for high confidence peptide identification. Again, the original mature N-terminal peptide presented CLIP-TRAQ reporter ions with equal intensities in the 113 (MMP-2) and the 114 (control) channels (Fig. 5B), whereas a second peptide assigned to the proEMAP/p43 sequence gave rise only to a reporter ion in the 113 channel (Fig. 5B). Therefore, the latter is a neo-N-terminus revealing MMP-2 cleavage of proEMAP/p43 at position 170 in the mature protein. Here too, high overall amino acid sequence homology and identity of the cleavage site sequence for the mouse and the human proEMAP/p43 proteins validated processing by MMP-2 for both species (supplemental Fig. 4B). Cleavage of proEMAP/p43 by MMP-2 was confirmed by SDS-PAGE analysis of recombinant murine proEMAP/p43 incubated with the protease (Fig. 5C) that could be inhibited by the broad spectrum MMP inhibitor Marimastat (60).


A statistics-based platform for quantitative N-terminome analysis and identification of protease cleavage products.

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

Processing of murine proEMAP/p43 by MMP-2. A, amino acid sequence of proEMAP/p43 showing the acetylated natural N-terminus of the mature protein (green) and an MMP-2-generated neo-N-terminus (blue) identified by CLIP-TRAQ-TAILS. B, mass spectra assigned to the natural N-terminus and neo-N-terminus of proEMAP/p43, respectively. Insets show a close-up of the CLIP-TRAQ reporter ion region indicating the presence of the natural N-terminal peptide in both samples and the neo-N-terminus only in the MMP-2-treated (CLIP-TRAQ-113) sample. C, MMP-2 cleavage of proEMAP/p43 analyzed by 15% Tris-Tricine SDS-PAGE. Arrows show full-length and cleavage products of proEMAP/p43.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Processing of murine proEMAP/p43 by MMP-2. A, amino acid sequence of proEMAP/p43 showing the acetylated natural N-terminus of the mature protein (green) and an MMP-2-generated neo-N-terminus (blue) identified by CLIP-TRAQ-TAILS. B, mass spectra assigned to the natural N-terminus and neo-N-terminus of proEMAP/p43, respectively. Insets show a close-up of the CLIP-TRAQ reporter ion region indicating the presence of the natural N-terminal peptide in both samples and the neo-N-terminus only in the MMP-2-treated (CLIP-TRAQ-113) sample. C, MMP-2 cleavage of proEMAP/p43 analyzed by 15% Tris-Tricine SDS-PAGE. Arrows show full-length and cleavage products of proEMAP/p43.
Mentions: As a second example, proEMAP/p43, a member of the small inducible cytokine family, was identified by an acetylated N-terminus after removal of the initiator methionine (Fig. 5, A and B). Although the corresponding Swiss-Prot entry (P31230) lacks this information, it was also predicted by the TermiNator algorithm (58) with 83% confidence. Importantly, the TAILS negative selection strategy allows the identification of acetylated N-termini that are proposed for up to 70% of all proteins (59) that positive selection strategies cannot detect. In addition, isotopic labeling of lysine side chains preserves quantification data provided the naturally blocked N-terminus harbors a lysine in its sequence as demonstrated for this example. Because CLIP-TRAQ labeling is at the protein level with trypsinization performed afterward, cleavage only occurs at arginine residues, preserving the label and increasing the size of the peptide and thus the probability for high confidence peptide identification. Again, the original mature N-terminal peptide presented CLIP-TRAQ reporter ions with equal intensities in the 113 (MMP-2) and the 114 (control) channels (Fig. 5B), whereas a second peptide assigned to the proEMAP/p43 sequence gave rise only to a reporter ion in the 113 channel (Fig. 5B). Therefore, the latter is a neo-N-terminus revealing MMP-2 cleavage of proEMAP/p43 at position 170 in the mature protein. Here too, high overall amino acid sequence homology and identity of the cleavage site sequence for the mouse and the human proEMAP/p43 proteins validated processing by MMP-2 for both species (supplemental Fig. 4B). Cleavage of proEMAP/p43 by MMP-2 was confirmed by SDS-PAGE analysis of recombinant murine proEMAP/p43 incubated with the protease (Fig. 5C) that could be inhibited by the broad spectrum MMP inhibitor Marimastat (60).

Bottom Line: Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode.The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF).By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed.

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
Terminal amine isotopic labeling of substrates (TAILS), our recently introduced platform for quantitative N-terminome analysis, enables wide dynamic range identification of original mature protein N-termini and protease cleavage products. Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode. The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF). Furthermore, the isoform assignment score (IAS) is introduced, a new scoring system for the evaluation of single peptide-to-protein assignments based on high confidence protein identifications in the same sample prior to negative selection enrichment of N-terminal peptides. By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed.

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