Identification of protease substrates by combinatorial profiling on TentaGel beads

Kofoed, Jacob; Reymond, Jean‐Louis

doi:10.1039/b713595e

Cited by 12 publications

(11 citation statements)

References 35 publications

(10 reference statements)

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“…The limitation of the proposed method is that lysine and arginine cannot be included in library sequences, since their side chains give color products with ninhydrin. Recently, Kofoed and Reymond [24] proposed an alternative selective labeling of the free N-terminus by reductive alkylation with 4-carboxybenzaldehyde coupled to Disperse Red 1 in the presence of NaBH 3 CN. The reagent can selectively stain-free amino groups liberated by proteolysis of peptides on TentaGel beads, even in presence of guanidine group of arginine residue.…”

Section: Application Of Qas Containing Linker To Oboc Librarymentioning

confidence: 99%

Sensitive electrospray mass spectrometry analysis of one-bead-one-compound peptide libraries labeled by quaternary ammonium salts

et al. 2012

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A rapid and straightforward method for highthroughput analysis of single resin beads from one-beadone-compound combinatorial libraries with high resolution electrospray ionization tandem mass spectrometry (HR ESI-MS/MS) is presented. The application of an efficient method of peptide derivatization by quaternary ammonium salts (QAS) formation increases ionization efficiency and reduces the detection limit, allowing analysis of trace amounts of compounds by ESI-MS. Peptides, synthesized on solid support, contain a new cleavable linker composed of a Peg spacer (9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid), lysine with ε-amino group marked by the N,N,N-triethylglycine salt, and methionine, which makes possible the selective cleavage by cyanogen bromide. Even a small portion of peptides derivatized by QAS cleaved from a single resin bead is sufficient for sequencing by HR ESI-MS/MS experiments. The developed strategy was applied to a small training library of α chymotrypsin substrates. The obtained results confirm the applicability of the proposed method in combinatorial chemistry. Keywords Derivatization of peptides · ESI-MS/MS

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Section: Application Of Qas Containing Linker To Oboc Librarymentioning

confidence: 99%

Sensitive electrospray mass spectrometry analysis of one-bead-one-compound peptide libraries labeled by quaternary ammonium salts

et al. 2012

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“…[18] In this assay the free amino termini unmasked by proteolysis are derivatized by reductive alkylation with the dye-labeled aldehyde 1, which reveals proteolysis on the surface of the solid support known as "bead-shaving".…”

Section: Resultsmentioning

confidence: 99%

“…A 4096-member combinatorial library was prepared with variable amino acid positions in the first and second generation tripeptide branches following our previously reported library design protocol (Figure 2). [17] While no reaction could be detected with trypsin or a-chymotrypsin under the conditions of protease profiling with linear peptides (25 8C, 1 h in aqueous buffer), [18] incubation at 36 8C in the presence of dimethylsulfoxide (20 %, v/v) as cosolvent provided a significant proportion of stained beads after reductive alkylation. In both cases, the sequences of protease reactive (stained) and unreactive (unstained) beads were analyzed (Table S1-S5 in the Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Proteolysis of Peptide Dendrimers

et al. 2009

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The ability of proteins and peptides to undergo proteolysis is essential to their biological function. Herein, we report the first detailed study of the protease reactivity of peptide dendrimers. Dendrimers are regularly ramified, tree-like synthetic macromolecules with promising application in technology and medicine. Using trypsin and alpha-chymotrypsin cleavage sites as models, we show that the protease reactivity of peptide dendrimers can be controlled by the degree of branching. Dendrimers with two or three amino acids between branching points were readily cleaved by trypsin irrespective of the position of the reactive sequence within the dendrimers, for example in D1, (Ac-Gly-Phe-Pro)4(Dap-Hyp-Arg[downward arrow]Met)2Dap-Ser-Gly-betaAla-NH2, and D12, (Ac-Ser-Ala)8(Dap-Ala-Arg[downward arrow])4(Dap-Ala-Asp)2Dap-Phe-Ala-Lys*-NH2 (Dap: (S)-2,3-diaminopropionic acid branching point, Hyp: hydroxyproline, Lys*: FITC-labeled lysine, [downward arrow]: cleavage site). On the other hand cleavage was blocked in more compact dendrimers with only one amino acid between branching points, for example in D18B, (Ac-Glu)8(Dap-Phe)4(Dap-Arg)2Dap-Leu-NH2). The control of proteolysis by topology provides a novel possibility to tune the biological properties of peptide dendrimers not available in linear peptides, and should be generally useful for their use as functional biomolecule analogues, for example, in the context of drug delivery applications.

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“…For the case of proteases combinatorial assays are particularly in demand for testing multiple peptides in parallel and determining the cleavage specifi city [54] . New solutions to this problem using cocktails of fl uorescence -labeled peptides [55] or combinatorial peptide libraries were recently reported [56,57] .…”

Section: Amidases and Proteasesmentioning

confidence: 99%