Peptide Microarrays for the Determination of Protease Substrate Specificity

Salisbury, Cleo M.; Maly, Dustin J.; Ellman, Jonathan A.

doi:10.1021/ja027477q

Cited by 251 publications

(182 citation statements)

References 28 publications

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“…Methods-The fluorogenic substrate library was synthesized and printed according to protocols previously described (5,6,9). The P 1 ϭ Arg and P 1 ϭ Lys sublibraries, along with calibration standards (unacylated 7-amino-4-carbamoylmethylcoumarin (ACC), acetylcapped ACC, and blanks), were printed at either 50 or 100 M in a 16 ϫ 24 format equivalent to a 384-well plate on polylysine-coated glass slides (Erie Scientific, Portsmouth, NH) using a 1 ϫ 1 pin (Telechem, Sunnyvale, CA) protocol on an OmniGrid Accent (Gene Machines, San Carlos, CA) microarrayer.…”

mentioning

confidence: 99%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

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151

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Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format AcAla-X-X-(Arg/Lys)-coumarin was synthesized (X ‫؍‬ all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXa␤, factor XIa and factor ␣ XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates ؋ 24 different proteases ؋ 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P 2 preference for Leu, Val, Phe, and Tyr in both the P 1 ‫؍‬ Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage. Molecular & Cellular Proteomics 4:626 -636, 2005.Because of their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases regulate hormonal activation, cellular homeostasis, apoptosis, and coagulation and play an important role in the pathogenicity and progression of many diseases (1). Proteases comprise one of the largest protein families in organisms from Escherichia coli to humans (2-4). Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets (1).To properly function, proteases must preferentially cleave their target substrates in the presence of other proteins. While many factors impact protease substrate selection, one of the key aspects is the complementary nature of the enzymeactive site with the residues surrounding the cleaved bond in the substrate. As such, determination of the residues that comprise the preferred cleavage site of a protease provides critical information regarding substrate selection. Furthermore, determination of substrate specificity also provides a framework for the design of potent and selective inhibitors.Here we exploit solution-phase substrate nanodroplet microarrays (5), in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles (6). These arrays allow high throughput characterization of the preferred residues on the P side (7) of the substrate in a highl...

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confidence: 99%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

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151

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“…An entire positional protease substrate library (25) with 19 different amino acids in the P2 through P4 positions (6,859 compounds) can be accommodated on a microarray. Recent work has begun to link fluorogenic peptide substrates to microarrays where the peptide group is released on proteolysis resulting in a fluorescent position on the microarray (26). Our preliminary studies with a 361-compound Ac-A-P3-P2-K-ACC peptide library arrayed in glycerol buffer and activated with deposited thrombin (Ϸ250 nM thrombin in the reaction) showed a strict requirement for proline in the P2 position and gave very similar results for P3 specificity as found by well plate and slide-based assay (24)(25)(26).…”

Section: Discussionmentioning

confidence: 99%

Printing chemical libraries on microarrays for fluid phase nanoliter reactions

Gosalia

Diamond

2003

Proc. Natl. Acad. Sci. U.S.A.

139

110

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Chemical compounds within individual nanoliter droplets of glycerol were microarrayed onto glass slides at 400 spots͞cm 2 . Using aerosol deposition, subsequent reagents and water were metered into each reaction center to rapidly assemble diverse multicomponent reactions without crosscontamination or the need for surface linkage. This proteomics technique allowed the kinetic profiling of protease mixtures, protease-substrate interactions, and highthroughput screening reactions. An inhibitor of caspases 2, 4, and 6 was identified by using a 352-compound combinatorial library microarrayed in quadruplicates on 100 slides and screened against caspases 2, 4, and 6, as well as thrombin and chymotrypsin. From one printing run that consumes <1 nanomole of each compound, large combinatorial libraries can be subjected to numerous separation-free homogeneous assays at volumes 10 3 -10 4 smaller than current high-throughput methods. DNA chips and microarrays for genotyping and expression profiling give no information about the activities of enzymes that can be regulated by posttranslational modifications or cleavage state. Protein microarrays have used the capture of proteins to libraries of immobilized DNA sequences, peptides, antibodies, chemical motifs, or other proteins(1-6). The three major formats for protein arrays currently use plain glass slides (1), 3D gel pad chips (7) (''matrix'' chips), or nanowell chips (2, 3). All of the formats require pretreatment for immobilization, which is a laborious and potentially protein-altering process. Also, none of these three formats have used soluble fluorogenic substrates to quantify multicomponent reactions common to high-throughput screening or to quantify numerous enzymes in a sample like plasma. Microarray-based activity assays of kinases require the use of immobilized substrates (2, 6) and cannot be easily implemented for the direct screening of soluble compounds from a combinatorial library. Assays performed in well plates before arraying onto chips for detection do not exploit the scale down or liquid handling power of microarray printing (6). Thus, a need exists to create localized reaction volumes in an array-based format as well as to create a method of rapidly delivering small volumes of fluid to each reaction. Additionally, evaporation effects typically prevent the extreme scale down of well-plate reactions to nanoliter volumes (8). We have developed a technique that performs enzymatic assays at nanoliter volumes in the liquid phase with minimal evaporation, no crosscontamination, and high reproducibility, and that has the capability to rapidly assemble multicomponent reactions with minimal sample usage in a microarray format. Methods. An OmniGrid Accent and OmniGrid contact microarrayers (Gene Machines, San Carlos, CA) were used for arraying. Arrays were printed on the OmniGrid Accent by using a 1 ϫ 1 pin protocol or on the OmniGrid with a 4 ϫ 8 pin protocol. All glass slides (Erie Scientific, Portsmouth, NH) were washed in dry ethanol and vacuum dried before arra...

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“…98,108 In an effort to understand the enzymatic pathways and functions, substrate profiling of protein kinase, phosphatase and protease has been performed on peptide microarrays by printing or synthesizing peptide-based molecules on surfaces ( Figure 18). 99,[112][113][114][115] More importantly, substrate specificity determination greatly aids the design of novel enzyme substrates and inhibitors. 113 It is possible to identify new enzyme inhibitors and evaluate their IC 50 values using peptide-or peptoid-arrays.…”

Section: Discovery Of Ligands On Microarraymentioning

confidence: 99%

“…99,[112][113][114][115] More importantly, substrate specificity determination greatly aids the design of novel enzyme substrates and inhibitors. 113 It is possible to identify new enzyme inhibitors and evaluate their IC 50 values using peptide-or peptoid-arrays. 90,116 In Figure 19, activity-based protein profiling (ABPP), which utilizes active-site-directed probes to profile the functional states of enzymes in proteomes, has also been performed on microarrays with improvements in sensitivity, resolution and enzyme identification as compared to the traditional liquid chromatography-mass spectrometry (LC-MS) method.…”

Section: Discovery Of Ligands On Microarraymentioning

confidence: 99%

Exploring Peptide Space for Enzyme Modulators

Cai

Johnston

et al. 2010

J. Am. Chem. Soc.

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Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry.Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis.Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening highdensity arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and β-galactosidase (β-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays.Several peptides, selected from microarrays, were able to inhibit β-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation.PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover ii molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry.

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Peptide Microarrays for the Determination of Protease Substrate Specificity

Cited by 251 publications

References 28 publications

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Printing chemical libraries on microarrays for fluid phase nanoliter reactions

Exploring Peptide Space for Enzyme Modulators

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