A high-complexity, multiplexed solution-phase assay for profiling protease activity on microarrays

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 Bottom‐Up Proteomic Approach to Identify Substrate Specificity of Outer‐Membrane Protease OmpT

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer‐membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1′) and nearest‐neighbor positions (P2, P2′) and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400‐fold improvement in OmpT catalytic efficiency, with a k _cat/K _m value of 6.1×10⁶ L mol⁻¹ s⁻¹. Wild‐type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.     

Endo- and aminopeptidase activities of rat cathepsin H.

Employing soluble denatured protein substrates and their derivatives, the proteolytic activity of rat cathepsin H was investigated. The enzyme showed aminopeptidase activity which sequentially released amino acid from the N-terminal of the substrate. The aminopeptidase activity did not act on N alpha-acetylated peptides and showed moderate ionic-strength dependence when methionyl-methylcoumarylamide was employed as a substrate. These results indicate that the activity essentially requires an N-terminal free amino group of the substrate and recognizes it electrostatically to some extent. On the other hand, the enzyme was also indicated to exhibit endopeptidase activity by employing appropriate N alpha-acetylated peptide substrates. In contrast to the aminopeptidase activity, the endopeptidase activity showed rather strict specificity, preferring hydrophobic residues at P2 and P3 sites. Because of the broad specificity and high efficiency of the aminopeptidase activity, it was difficult to directly observe endopeptidase activity in the digestion of large peptide substrates with a free alpha-amino terminal. Thus, this is the first experimental evidence that indicates endopeptidase activity by assigning internal peptide bonds cleaved by this activity. From this data, we proposed a model of the binding site of this enzyme.     

Identification of photolabeled peptides for the acceptor substrate binding domain of beta 1,4-galactosyltransferase.

We successfully applied a carbene-generating N-acetylglucosamine derivative carrying a biotinyl group to the radioisotope-free identification of peptides within bovine UDP-galactose: N-acetylglucosamine beta 1,4-galactosyltransferase (GalT, EC 2.4.1.38) catalytic domain. Owing to the low yield of cross-linking, conventional photoaffinity labeling experiments usually encounter a thorny problem in attempting to isolate labeled components from very complex mixtures. A biotin tag introduced with our photoaffinity probe enabled us to separate the photolabeled protein from a large amount of coexisting unlabeled GalT. The introduction of biotin was also useful for the radioisotope-free detection of a labeled protein based on a highly sensitive chemiluminescent technique. We developed a novel poly(vinylidene difluoride) membrane for the identification of labeled peptides in a simple dot blot assay. Using this membrane, we successfully identified biotinyl peptides among a number of HPLC separated fragments derived from the protease digestion of photolabeled GalT proteins. The sequence analysis revealed that the biotin tag was incorporated within a tryptic GalT fragment of Y197-R208. Our approach yields, for the first time, information on the acceptor substrate binding-site fragment in this enzyme, that has been difficult to obtain using other approaches. These data are consistent with previous suggestions concerning the GalT acceptor site and clearly demonstrate the effectiveness of our approach for rapid identification of photolabeled peptides.     

Detection of protease activities using specific aminoacyl or peptidyl p-nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications

A general method for detecting protease activities on acrylamide or agarose gels after sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) using specific aminoacyl p-nitroanilide (NA) or peptidyl NA as substrate is described. This method is extended from the spectrophotometric assay of p-nitroaniline, which is a chromogenic product liberated by protease action on aminoacyl NA or peptidyl NA. The acrylamide gel containing protein bands was dipped directly into a solution which contained specific synthetic aminoacyl NA or peptidyl NA as a substrate or had been overlaid with an agarose gel containing the same substrate. The p-nitroaniline released on the acrylamide or agarose gel by the specific protease was diazotized with sodium nitrite and then coupled to N-(1-naphthyl)-ethylenediamine to produce distinct activity band(s). The substrates used for protease activity staining on gels were identical to those used for spectrophotometric assays. Some applications are described.     

An Efficient Method for the In Vitro Production of Azol(in)e‐Based Cyclic Peptides

Heterocycle‐containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine‐derived enzymes and substrates obtained from a family of ribosomally produced and post‐translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non‐native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6–9 residues representing 11 out of the 20 canonical amino acids.     

Reverse zymography using fluorogenic substrates for protease inhibitor detection

A novel, sensitive method for detecting protease inhibitors by using fluorescent protease substrates in gels is described. The protease inhibitors were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels containing a copolymerized peptide substrate, namely 4-methyl-coumaryl-7-amide (MCA). As the incorporated substrates in the gel, Boc-Phe Ser-Arg-MCA was used for trypsin, Suc-Ala-Ala-Pro-Phe-MCA for alpha-chymotrypsin, and Z-Phe-Arg-MCA for papain. After electrophoresis, washing and incubating the gel with the target protease solutions allowed the substrate to be cleaved by the protease, and the release of the fluorescent 7 amino-4 methyl-coumarin (AMC), which was detected under a UV transilluminator. The uncleaved peptide-MCA substrate remained where the inhibitors were present, and was visualized as dark blue bands on the light-green fluorescent background gel. This new method offers several advantages over other previous methods including: (i) greatly increased sensitivity can be achieved in a shorter period of time, which may be useful for discovering new protease inhibitors in small amounts of crude material; (ii) the procedure is quite simple and quick since the incubation period is very short and no time is needed for staining and destaining steps; (iii) since these probes using substrate specificity/target proteases, they are excellent tools for detection and discrimination of unknown protease inhibitors for various target proteases.     

Aryldithioethyloxycarbonyl (Ardec): a new family of amine protecting groups removable under mild reducing conditions and their applications to peptide synthesis

The development of phenyldithioethyloxycarbonyl (Phdec) and 2-pyridyldithioethyloxycarbonyl (Pydec) protecting groups, which are thiol-labile urethanes, is described. These new disulfide-based protecting groups were introduced onto the epsilon-amino group of L-lysine; the resulting amino acid derivatives were easily converted into N alpha-Fmoc building blocks suitable for both solid- and solution-phase peptide synthesis. Model dipeptide(Ardec)s were prepared by using classical peptide couplings followed by standard deprotection protocols. They were used to optimize the conditions for complete thiolytic removal of the Ardec groups both in aqueous and organic media. Phdec and Pydec were found to be cleaved within 15 to 30 min under mild reducing conditions: i) by treatment with dithiothreitol or beta-mercaptoethanol in Tris.HCl buffer (pH 8.5-9.0) for deprotection in water and ii) by treatment with beta-mercaptoethanol and 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) in N-methylpyrrolidinone for deprotection in an organic medium. Successful solid-phase synthesis of hexapeptides Ac-Lys-Asp-Glu-Val-Asp-Lys(Ardec)-NH2 has clearly demonstrated the full orthogonality of these new amino protecting groups with Fmoc and Boc protections. The utility of the Ardec orthogonal deprotection strategy for site-specific chemical modification of peptides bearing several amino groups was illustrated firstly by the preparation of a fluorogenic substrate for caspase-3 protease containing the cyanine dyes Cy 3.0 and Cy 5.0 as FRET donor/acceptor pair, and by solid-phase synthesis of an hexapeptide bearing a single biotin reporter group.     

Carbon Nanotube‐Enhanced Polarization of Fluorescent Peptides: A Novel Amplification Strategy for Homogeneous Detection of Proteases

A new fluorescence polarization (FP) amplification strategy based on the use of multiwalled carbon nanotubes (MWCNTs) as the FP enhancer was developed for the simple, sensitive, and universal monitoring of protease activity in homogeneous solution. A fluorophore‐labeled peptide that includes a protease‐cleavable element and ten histidine residues for binding MWCNTs is adsorbed on MWCNTs through strong π–π stacking and electrostatic interactions. When the fluorophore‐labeled peptide/MWCNT complexes are exposed to a protease target, specific peptide cleavage by the protease target occurs, thus releasing fragments carrying the fluorophore from the surface of MWCNTs, which in turn results in a significant decrease in the FP value. The detection limits of this assay for two proteases, thrombin and chymotrypsin (CTP), were estimated to be 0.5 pM and 0.3 pM , respectively. In addition, it is also demonstrated that this MWCNT‐enhanced FP assay is suitable for protease inhibitor screening.     

Identification of calpain substrates by ORF phage display

Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases.     

Electrochemical proteolytic beacon for detection of matrix metalloproteinase activities

This communication describes a novel method for detecting matrix metalloproteinase-7 activity using a peptide substrate labeled with a ferrocene reporter. The substrate serves as a selective "electrochemical proteolytic beacon" (EPB) for this metalloproteinase. The EPB is immobilized on a gold electrode surface to enable "on-off" electrochemical signaling capability for uncleaved and cleaved events. The EPB is efficiently and selectively cleaved by MMP-7 as measured by the rate of decrease in redox current of ferrocene. Direct transduction of a signal corresponding to peptide cleavage events into an electronic signal thus provides a simple, sensitive route for detecting the MMP activity. The new method allows for identification of the activity of MMP-7 in concentrations as low as 3.4 pM. The concept can be extended to design a multiple peptide substrate labeled with different electroactive reporters for assaying multiple MMPs activities.     

Assay and Kinetic Studies on the Enzymatic Proteolysis with Ninhydrin Reagent

A method using ninhydrin reagent for proteolytic activity measurement and kinetic studies with protein or modified protein as substrate was explored. As the reagent is specific for amino group, quantitative expression can be made with the number of peptide bonds cleaved. The sensitivity of the method made it possible for the determination of various kinetic parameters such as K_m, V_max and K_cat with the measurement of initial velocity. As an example, these quantities were determined for pancreas protease with casein and N,N-dimethylcasein as substrates. This study made clear the possibility of using a common substrate of proteolytic enzymes for kinetic studies and a general reference for activity expression. By introducing the use of initial velocity in the kinetic studies of proteolysis with protein as the substrate, this study also makes clear that the kinetics of the enzymatic proteolysis very well fits the Michaelis-Menten law instead of the Schütz law.     

A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon—carbon bonds in cysteine.     

Inhibition of hepatitis C virus serine protease in living cells by RNA aptamers detected using fluorescent protein substrates

Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.     

Macrocyclic Protease Inhibitors with Reduced Peptide Character

There is a real need for simple structures that define a β‐strand conformation, a secondary structure that is central to peptide–protein interactions. For example, protease substrates and inhibitors almost universally adopt this geometry on active site binding. A planar pyrrole is used to replace two amino acids of a peptide backbone to generate a simple macrocycle that retains the required geometry for active site binding. The resulting β‐strand templates have reduced peptide character and provide potent protease inhibitors with the attachment of an appropriate amino aldehyde to the C‐terminus. Picomolar inhibitors of cathepsin L and S are reported and the mode of binding of one example to the model protease chymotrypsin is defined by X‐ray crystallography.     

2‐Oxazoline Formation for Selective Chemical Labeling of 5‐Hydroxylysine

Hydroxylation of lysine, one of posttranslational modifications of proteins, generates 5‐hydroxylysine (Koh) and plays a crucial role in regulating protein functions in cellular activity. We have developed a chemical labeling method of Koh. The 1,2‐aminoalcohol moiety of Koh in synthetic peptide sequences was trapped by an alkyne‐containing benzimidate to form a 2‐oxazoline ring. An additional ammonia treatment process removed the undesirable amidine residue formed between benzimidate and lysine. During the ammonia treatment, the oxazoline residue formed at Koh mainly remained intact, and the ring opening to the amide form was observed for only part of oxazoline, indicating that the chemical labeling is amino acid selective. Azide‐substituted biotin or fluorescent dye was attached to the peptide through Huisgen cycloaddition at Koh and converted into an alkyne‐labeled oxazoline form. The Koh‐labeling assay could provide a platform to enhance proteomic research of lysine hydroxylation.     

Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy.

Transmissible spongiform encephalopathies in humans and in animals are fatal neuro-degenerative diseases with long incubation times. The putative cause of these diseases is a normal host protein, the prion protein, that becomes altered. This abnormal prion protein is found mostly in the brains of infected individuals in later stages of the disease, but also can be found in lymphoid and other tissues in lower amounts. In order to eradicate this disease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensitivity that can be achieved with capillary electrophoresis makes it possible to detect the abnormal protein in blood. A peptide from the carboxyl terminal region, amino acid positions 218-232, was labeled with fluorescein during the synthesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillary electrophoresis immunoassay. The amount of fluorescein labeled peptide in the capillary was 50 amol. Blood was obtained from normal sheep and elk, from sheep infected with scrapie and elk infected with chronic wasting disease. Buffy coats and plasma were prepared by a conventional method. After treatment with proteinase K, which destroys the normal protein but not the altered one, the blood fractions were extracted and tested in the capillary electrophoresis immunoassay for the abnormal prion protein. The abnormal prion protein was detected in fractions from blood from infected animals but not from normal animals. This assay makes a pre-clinical assay possible for these diseases and could be adapted to test for the abnormal prion protein in process materials that are used for manufacture of pharmaceuticals and products for human consumption.     

Outer-Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate

E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFU mL⁻¹ of E. coli in water within 6 hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37_FRRV) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37_FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37_FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37_FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37_FRRV sequence variants.