A High-Complexity, Multiplexed Solution-Phase Assay for Profiling Protease Activity on Microarrays

Abstract

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.

A Novel Continuous Assay for the Deacylase Sirtuin 5 and Other Deacetylases

“…Obviously, these modifications of 1 did not negatively influence the substrate properties for Sirt5 as shown in Figure . The helper protease trypsin disfavors negatively charged side chains in P1′-position, leading to high K M value for 4b , the product of the Sirt5 mediated desuccinylation of 4a (Figure S1). Therefore, we exchanged the glutamate by an alanine leading to the derivatives 5a and 5b .…”

“…H3 K9-Succ negatively charged side chains in P1′-position, 37 leading to high K M value for 4b, the product of the Sirt5 mediated desuccinylation of 4a (Figure S1). Therefore, we exchanged the glutamate by an alanine leading to the derivatives 5a and 5b.…”