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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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Related in: MedlinePlus

MMP-2 active site mapping. A, heat map for the amino acid occurrences in P4–P4′ for all identified MMP-2-generated neo-N-termini (n = 1,183). CLIP-TRAQ-TAILS analysis confirms the predominant MMP-2 preferences for leucine in P1′ and proline in P3 position. B, protein sequence logo calculated from the same data set applying correction for natural amino acid abundance. The logo was generated using the iceLogo software package (48).
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Figure 6: MMP-2 active site mapping. A, heat map for the amino acid occurrences in P4–P4′ for all identified MMP-2-generated neo-N-termini (n = 1,183). CLIP-TRAQ-TAILS analysis confirms the predominant MMP-2 preferences for leucine in P1′ and proline in P3 position. B, protein sequence logo calculated from the same data set applying correction for natural amino acid abundance. The logo was generated using the iceLogo software package (48).

Mentions: Because of the large number of cleavage events identified by CLIP-TRAQ-TAILS analysis, it can also be used to map protease active sites using native protein rather than peptide substrates (47, 61). We derived consensus sequences for 4 amino acids upstream of the identified cleavage site referred to as P4 to P1 (62) for all 1,183 cleavage events by mapping to all matching isoforms as described previously (47). Unambiguous prime side amino acids (P1′ to P4′) were derived from the actual neo-N-terminal peptide sequences. As indicated by the heat map (Fig. 6A) and protein sequence logo analysis (Fig. 6B) of the active site, our results are in very good agreement with a previous study using proteome-derived peptide libraries (47). Most prominent are the MMP characteristic preferences for proline in P3 (21%) and leucine in P1′ (40%) positions. In addition, we identified the same preference for alanine (19%), glycine (12%), and serine (14%) in P2; alanine (14%) and glycine (12%) in P1; and alanine (16%), glycine (14%), and serine (15%) in P3′ positions, adding further weight to their being bona fide cleavage sites.


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)

MMP-2 active site mapping. A, heat map for the amino acid occurrences in P4–P4′ for all identified MMP-2-generated neo-N-termini (n = 1,183). CLIP-TRAQ-TAILS analysis confirms the predominant MMP-2 preferences for leucine in P1′ and proline in P3 position. B, protein sequence logo calculated from the same data set applying correction for natural amino acid abundance. The logo was generated using the iceLogo software package (48).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871423&req=5

Figure 6: MMP-2 active site mapping. A, heat map for the amino acid occurrences in P4–P4′ for all identified MMP-2-generated neo-N-termini (n = 1,183). CLIP-TRAQ-TAILS analysis confirms the predominant MMP-2 preferences for leucine in P1′ and proline in P3 position. B, protein sequence logo calculated from the same data set applying correction for natural amino acid abundance. The logo was generated using the iceLogo software package (48).
Mentions: Because of the large number of cleavage events identified by CLIP-TRAQ-TAILS analysis, it can also be used to map protease active sites using native protein rather than peptide substrates (47, 61). We derived consensus sequences for 4 amino acids upstream of the identified cleavage site referred to as P4 to P1 (62) for all 1,183 cleavage events by mapping to all matching isoforms as described previously (47). Unambiguous prime side amino acids (P1′ to P4′) were derived from the actual neo-N-terminal peptide sequences. As indicated by the heat map (Fig. 6A) and protein sequence logo analysis (Fig. 6B) of the active site, our results are in very good agreement with a previous study using proteome-derived peptide libraries (47). Most prominent are the MMP characteristic preferences for proline in P3 (21%) and leucine in P1′ (40%) positions. In addition, we identified the same preference for alanine (19%), glycine (12%), and serine (14%) in P2; alanine (14%) and glycine (12%) in P1; and alanine (16%), glycine (14%), and serine (15%) in P3′ positions, adding further weight to their being bona fide cleavage sites.

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.

Show MeSH
Related in: MedlinePlus