Dual colour, microarray-based, analysis of 10 000 protease substrates

Díaz‐Mochón, Juan José; Bialy, Laurent; Bradley, Mark

doi:10.1039/b609029j

Cited by 52 publications

(37 citation statements)

References 17 publications

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“…6 Detection of proteolytic cleavage on peptide arrays reading surface-bound fragment. a Internally fluorescence-quenched peptides on arrays, subsequent to cleavage peptide fragment containing quenching moiety is washed away yielding fluorescence increase for cleaved peptides [66,166,171]. b Peptides having a fluorogenic group C-terminal to the scissile bond which increases fluorescence subsequent to cleavage [175,181].…”

Section: Assays and Detectionmentioning

confidence: 99%

“…Additionally, for protein-tyrosine kinase p60 c-src only incorporation of the long and hydrophilic 1-amino-4,7,10-trioxa-13-tridecanamine succinimic acid building block spacer allowed effective phosphorylation of the glass surface-bound peptides [26]. A similar linker structure was used to space apart Peptide Nucleic Acid (PNA) tags from potential protease substrates [66], protein serine/threonine kinase substrates [29,30] or tyrosine kinase substrates [67]. Inamori et al reported that insertion of a PEG spacer between chemoselective attachment point and the kinase substrate sequence improved phosphorylation efficiency by c-Src but not by PKA [35].…”

Section: Chemistry Of Peptide Array Preparationmentioning

confidence: 99%

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Deciphering Enzyme Function Using Peptide Arrays

2011

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Enzymes are key molecules in signal-transduction pathways. However, only a small fraction of more than 500 human kinases, 300 human proteases and 200 human phosphatases is characterised so far. Peptide microarray based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimisation of enzyme substrates. Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases. Additionally, several examples for reliable identification of substrates for lysine methyl-transferases, histone deacetylases and SUMO-transferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples like cell lysates or lysates of complete organisms is described. All published examples of peptide arrays used for enzyme profiling are summarised comprehensively.

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Section: Assays and Detectionmentioning

confidence: 99%

Section: Chemistry Of Peptide Array Preparationmentioning

confidence: 99%

Deciphering Enzyme Function Using Peptide Arrays

2011

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“…Other types of soluble peptide libraries were prepared by individual coupling of peptides with DNA [232,233] or peptide nucleic acid [234] tags. After biotransformation of the peptide library in the solution assay (cleavage by protease, phosphorylation by kinase), the modified substrate was captured on a complementary DNA array and the structure of the substrate peptide was defined.…”

Section: Synthesis Of Peptide Mixturesmentioning

confidence: 99%

Combinatorial/Library Peptide Synthesis

Lebl

2011

Amino Acids, Peptides and Proteins in Organic Chemistry

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“…Such an approach has been applied to protease-activity analysis by utilizing peptide nucleic acid (PNA) encoding, [12][13][14] for phosphatase-activity screening with activity-based probes by expression display, [15] and we have used a similar approach to develop a protease-activity assay (I. A. Kozlov, et al, unpublished results).…”

Section: Introductionmentioning

confidence: 99%

A Multiplexed Protein Kinase Assay

et al. 2007

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We report a novel protein kinase assay designed for high-throughput detection of one or many kinases in a complex mixture. A solution-phase phosphorylation reaction is performed on 900 different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation, phosphoserine, phosphothreonine, and phosphotyrosine are chemically labeled, and the substrates are hybridized to a microarray with oligonucleotides complementary to the tags to read out the phosphorylation state of each peptide. Because protein kinases act on more than one peptide sequence, each kinase can be characterized by a unique signature of phosphorylation activity on multiple substrates. Using this method, we determined signatures for 26 purified kinases and demonstrated that enzyme mixtures can be screened for activity and selectivity of inhibition.

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Dual colour, microarray-based, analysis of 10 000 protease substrates

Abstract: A 10,000 member PNA-encoded library of FRET based peptides was synthesised for global analysis of protease cleavage specificity; analysis was achieved using a DNA microarray and consumed minimal quantities of enzyme (60 pmole) and library (3.5 nmole).

Cited by 52 publications

References 17 publications

Deciphering Enzyme Function Using Peptide Arrays

Deciphering Enzyme Function Using Peptide Arrays

Combinatorial/Library Peptide Synthesis

A Multiplexed Protein Kinase Assay

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