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Results: 5

1.
Figure 3

Figure 3. Substrate profiling of an exopeptidase PRCP. From: Global Identification of Peptidase Specificity by Multiplex Substrate Profiling.

PRCP releases amino acids from the C-terminus of peptides when Pro, Ala or Nle are in the P1 position. The time, if any, that cleavage was first observed is illustrated by the length of each bar.

Anthony J. O’Donoghue, et al. Nat Methods. ;9(11):1095-1100.
2.
Figure 4

Figure 4. Specificity constants of individual substrates can be calculated from the MSP-MS assay. From: Global Identification of Peptidase Specificity by Multiplex Substrate Profiling.

(a) The precursor ion abundance of KARSAFAEnWPDHN (black) and cleaved product (grey) at four time intervals after addition of Granzyme B to the MSP-MS assay. The precursor ion abundance was calculated using Xcalibur. (b) Progress curves of product formation for peptides KHPLETVYAD and KARSAFAEnWPD. In each case, the parent substrates were completely hydrolyzed by Granzyme B within 1200 minutes, therefore the concentration of each product is known and kinetic values were subsequently calculated.

Anthony J. O’Donoghue, et al. Nat Methods. ;9(11):1095-1100.
3.
Figure 1

Figure 1. Design of a physiochemically diverse peptide library and development of a multiplex substrate assay. From: Global Identification of Peptidase Specificity by Multiplex Substrate Profiling.

(a) Design of a library of 14-mer peptides by accommodating all neighbor (XY) and near neighbor pairs (X*Y and X**Y) into a core decapeptide (unshaded residues). X and Y correspond to defined amino acids while * corresponds to a random amino acid. The termini (shaded residues) were generated using amino acid pairs (XY) selected from 11 pools of amino acids. ‘n’ corresponds to norleucine. (b) Illustration of the multiplex substrate profiling assay. A peptidase or mixture of peptidases is added to the peptides and aliquots are removed at multiple time intervals and quenched. Samples are injected into a LC-MS/MS system to detect time dependant appearance of cleavage products.

Anthony J. O’Donoghue, et al. Nat Methods. ;9(11):1095-1100.
4.
Figure 2

Figure 2. Validation of the multiplex substrate profiling technique using the aspartyl peptidase, Cathepsin E. From: Global Identification of Peptidase Specificity by Multiplex Substrate Profiling.

(a) Quantitative assessment of cleaved bonds at increasing time intervals. (b–c) iceLogos generated from amino acids that are enriched or de-enriched in the P4 to P4′ position Cathepsin E cleavage sites after incubation for 5 minutes (b) and 20 hours (c). Percent difference corresponds to the difference of amino acid frequency surrounding the Cathepsin E cleavage sites relative to the frequency of amino acids surrounding all peptide bonds in the library (n= 1612). In the iceLogo, “M” corresponds to norleucine. (d) A bar chart representing tetrapeptide sequences containing Phe in the third position (**F↓*) and the time that cleavage is first observed. All **F* motifs in the library are listed in Supplementary Table 4.

Anthony J. O’Donoghue, et al. Nat Methods. ;9(11):1095-1100.
5.
Figure 5

Figure 5. Class specific peptidase inhibitors can dissect the proteolytic signatures of biological samples. From: Global Identification of Peptidase Specificity by Multiplex Substrate Profiling.

Substrate signature of material from (a) uninfected and (b) Schistosoma mansoni infected fresh water snails after 240 minutes incubation with the substrate library. (c) Infected snail material was pre-treated with AlaAlaProPhe-CMK inhibitor prior to multiplex substrate profile assay. (d) Cleavage site dataset of two semi-pure preparations of S. mansonii cercarial elastases were combined to generate a substrate signature for this peptidase group. (e) Substrate signature of all cleavage sites observed within 1200 minutes using conditioned media from pancreatic ductal adenocarcinoma cells as a source of proteases. Media was pre-treated with (f) E-64, (g) 1,10 phenanthroline and (h) pepstatin and a substrate signature was generated of the remaining cleavage events. In all cases, only amino acids that are enriched or de-enriched are illustrated in the substrate signature and ‘M’ corresponds to norleucine.

Anthony J. O’Donoghue, et al. Nat Methods. ;9(11):1095-1100.

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