A Dual‐Response DNA Probe for Simultaneously Monitoring Enzymatic Activity and Environmental pH Using a Nanopore

Both protease overexpression and local pH changes are key signatures of cancer. However, the sensitive detection of protease activities and the accurate measurement of pH in a tumor environment remain challenging. Here, we develop a dual‐response DNA probe that can simultaneously monitor protease activities and measure the local pH by translocation through α‐hemolysin (αHL). The DNA probe bears a short peptide containing phenylalanine at a pre‐designed position. Enzymatic cleavage of the peptide either exposes or removes the N‐terminal phenylalanine that can form a complex with cucurbit[7]uril. Translocation of the DNA hybrid through αHL generates current signatures that can be used to quantify protease activities. Furthermore, the current signatures possess a pH‐dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNA probe may be further explored for simultaneously measuring multiple parameters of a complex system such as single cells in the future.     

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.     

Optimizing electrode-attached redox-peptide systems for kinetic characterization of protease action on immobilized substrates. Observation of dissimilar behavior of trypsin and thrombin enzymes

In this work, we experimentally address the issue of optimizing gold electrode attached ferrocene (Fc)-peptide systems for kinetic measurements of protease action. Considering human α-thrombin and bovine trypsin as proteases of interest, we show that the recurring problem of incomplete cleavage of the peptide layer by these enzymes can be solved by using ultraflat template-stripped gold, instead of polished polycrystalline gold, as the Fc-peptide bearing electrode material. We describe how these fragile surfaces can be mounted in a rotating disk configuration so that enzyme mass transfer no longer limits the overall measured cleavage kinetics. Finally, we demonstrate that, once the system has been optimized, in situ real-time cyclic voltammetry monitoring of the protease action can yield high-quality kinetic data, showing no sign of interfering effects. The cleavage progress curves then closely match the Langmuirian variation expected for a kinetically controlled surface process. Global fit of the progress curves yield accurate values of the peptide cleavage rate for both trypsin and thrombin. It is shown that, whereas trypsin action on the surface-attached peptide closely follows Michaelis-Menten kinetics, thrombin displays a specific and unexpected behavior characterized by a nearly enzyme-concentration-independent cleavage rate in the subnanomolar enzyme concentration range. The reason for this behavior has still to be clarified, but its occurrence may limit the sensitivity of thrombin sensors based on Fc-peptide layers.     

Discerning matrix-cluster peaks in matrix-assisted laser desorption/ionization time-of-flight mass spectra of dilute peptide mixtures

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is widely used for the analysis of peptide mixtures such as those resulting from protein digestion. Among several useful peptide matrices, alpha-cyano-4-hydroxycinnamic acid (4-HCCA) appears to be the most popular. This matrix does not generally give matrix-cluster peaks at the mass region covered by enzyme-digested peptides (i.e., m/z above approximately 500). However, when an analyte mixture is very dilute and/or the sample contains a large amount of salts, ion peaks from matrix clusters can be quite intense, compared to peptide peaks. This matrix-cluster interference becomes more pronounced as the amount of analyte decreases. In this paper, a simple scheme for matrix-cluster identification is reported. It is shown that matrix-cluster formation follows a systematic pattern, although the relative intensities of these cluster ions cannot be predicted. Discerning the matrix-cluster peaks from the peptide peaks is found to be critical in analyzing dilute peptide mixtures with both conventional and microspot MALDI-TOF techniques.     

Identification of proteinaceous binders used in artworks by MALDI-TOF mass spectrometry

Proper identification of proteinaceous binders in artworks is essential for specification of the painting technique and thus also for selection of the restoration method; moreover, it might be helpful for the authentication of the artwork. This paper is concerned with the optimisation of analysis of the proteinaceous binders contained in the colour layers of artworks. Within this study, we worked out a method for the preparation and analysis of solid samples from artworks using tryptic cleavage and subsequent analysis of the acquired peptide mixture by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. To make this approach rational and efficient, we created a database of commonly used binders (egg yolk, egg white, casein, milk, curd, whey, gelatine, and various types of animal glues); certain peaks in the mass spectra of these binders, formed by rich protein mixtures, were matched to amino acid sequences of the individual proteins that were found in the Internet database ExPASy; their cleavage was simulated by the program Mass-2.0-alpha4. The method developed was tested on model samples of ground layers prepared by an independent laboratory and then successfully applied to a real sample originating from a painting by Edvard Munch.     

Electrochemical oxidation and cleavage of peptides analyzed with on-line mass spectrometric detection

An on-line electrochemistry/electrospray mass spectrometry system (EC/MS) is described that allows fast analysis of the oxidation products of peptides. A range of peptides was oxidized in an electrochemical cell by application of a potential ramp from 0 to 1.5 V during passage of the sample. Electrochemical oxidation of peptides was found to occur readily when tyrosine was present. Tyrosine was found to be oxidized between 0.5 and 1.0 V to various oxidation products, including peptide fragments formed by hydrolysis at the C-terminal side of tyrosine. The results confirm earlier knowledge on the mechanisms and reaction products of chemical and electrochemical peptide oxidation. Methionine residues are also readily oxidized, but do not induce peptide cleavage. At potentials higher than about 1.1 V, additional oxidation products were observed in some peptides, including loss of 28 Da from the C-terminus and dimerization. The tyrosine-specific cleavage reaction suggests a possible use of the EC/MS system as an on-line protein digestion and peptide mapping system. In addition, the system can be used to distinguish phosphorylated from unphosphorylated tyrosine residues. Four forms of the ZAP-70 peptide ALGADDSYYTAR with both, either or neither tyrosine phosphorylated were subjected to a 0-1.5 V potential ramp. Oxidation of, and cleavage adjacent to, tyrosine was observed exclusively at unphosphorylated tyrosine residues.     

An inhibitor of sequence-specific proteolysis that targets the substrate rather than the enzyme

BACKGROUND: Traditional protease inhibitors target the active site of the enzyme. However, since most proteases act on multiple substrates, even the most specific protease inhibitors will affect the levels of a number of different proteins. However, if substrate-targeted inhibitors could be developed, much higher levels of specificity could be achieved. In theory, compounds that bind the cleavage site of a particular substrate could block its interaction with a protease without having any effect on the processing of other substrates of that protease. RESULTS: A model system is presented that demonstrates the feasibility of substrate-targeted inhibition of proteolysis. A peptide selected genetically to bind a 14-residue epitope that encompasses the cleavage site of human pro-IL-1beta was shown to inhibit interleukin-converting enzyme (ICE)-mediated proteolysis of model substrates containing the 14-mer target sequence. However, the peptide had no effect on the cleavage of other ICE substrates with different amino acids flanking the minimal cleavage site. CONCLUSIONS: This study demonstrates the feasibility of substrate-targeted inhibition of proteolysis. More potent compounds must be developed before substrate-targeted inhibitors can be used routinely. Nonetheless, this novel strategy for protease inhibition seems promising for the development of extremely selective molecules with which to manipulate the maturation of many important pro-hormones, -cytokines and -proteins.     

Microbore liquid chromatography coupled to a flow fast atom bombardment probe for the on-line detection of the Tyr-Pro cleavage of a nonapeptide by recombinant HIV-1 protease

The nonapeptide Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln has been reported as a model substrate for an aspartyl protease produced by the human immunodeficiency virus (HIV-1). Cleavage of this peptide at the Tyr-Pro linkage to produce tetra- and pentapeptide fragments is the basis of high-performance liquid chromatographic assays to detect HIV-1 protease activity. Confirmation of the cleavage site has been proved by using microbore liquid chromatography coupled to a dynamic fast atom bombardment interface. Comparison with fortified control incubates indicates that an approximate stoichiometric amount of the tetrapeptide was formed from the nonapeptide, confirming that the cleavage of the substrate by HIV-1 protease is both specific and quantitative.     

Chemical Modification of the N Termini of Unprotected Peptides for Semisynthesis of Modified Proteins by Utilizing a Hydrophilic Protecting Group

A simple and efficient strategy for the selective modification of the peptide N terminus with an unnatural amino acid is described. A peptide having a SUMO-HisTag-TEV sequence (SUMO: small ubiquitin-related modifier, TEV: tobacco etch virus) preceding the N terminus of the target peptide was designed. Recombinant expression in E. coli and subsequent SUMO protease cleavage yielded the HisTag-TEV-target peptide. Partial protection of the lysine side chains of this peptide with d -glucopyranosyloxycarbonyl and removal of the HisTag-TEV sequence by TEV protease yielded the partially protected peptide with a free N-terminal amine. Coupling of selenocysteine selectively at the N terminus and subsequent acidic deprotection of the carbohydrate protecting groups yielded a modified peptide that can be used for native chemical ligation (NCL). As a proof of concept, the modification of a longer recombinant peptide with selenocysteinylserine (GalNAc) at the N terminus was demonstrated.     

Fragmentation of peptides with intra-chain disulfide bonds in triple quadrupole mass spectrometry and its quantitative application to biological samples

A growing number of peptides are being used today in bioanalytical laboratories. Because of this, there is an increasing interest in the development of highly sensitive, specific and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays for the quantitative analysis of peptides in biological samples. Among the mass spectrometers previously used for peptide quantification, triple quadrupole mass spectrometers are generally not considered the instrument of choice. With this instrumentation, collision cascades or multiple fragmentations tend to generate multiple peaks that have weak intensities. This leads to a loss in detection sensitivity. However, in cases where immonium product ions were formed in abundance, it was found that peptide quantification succeeded. A common feature of these peptides is their intra-loop structure. To elucidate the usefulness of this feature in fragmentation, several peptide analytes with intra-chain disulfide bonds were investigated in this study, including a newly synthesized analog having a single amino acid substitution. The results presented here indicate that abrupt bond cleavage from the intra-loop structure of peptides could be one of the premises for intense immonium ion generation. In contrast, any preferential cleavage of peptide bonds (e.g., proline effect) that gives rise to a linearized sequence would break the intactness of the loop and prevent it from completely dissociating. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of peptides with intra-chain disulfide bonds in biological fluids.     

Facile identification of photocleavable reactive metabolites and oxidative stress biomarkers in proteins via mass spectrometry

Described herein is a method which combines bond selective fragmentation by photodissociation with online liquid chromatographic separation and mass spectrometric analysis. Photoexcitation of proteins or peptides with 266-nm light does not normally yield abundant fragmentation; however, incorporation of a suitable carbon-sulfur or carbon-halogen bond that is proximal to a chromophore allows access to direct dissociation pathways, resulting in homolytic cleavage of these bonds. Radicals generated through this process can cause further dissociation of the peptide backbone, which is useful for site specifically identifying the point of modification. Two specific applications of this technique for peptide analysis in model systems are presented: (1) identification of reactive metabolites which covalently modify cysteine residues, and (2) characterization of halogenated tyrosine residues which are biomarkers related to oxidative stress. In both cases, these naturally occurring post translational modifications create photocleavable bonds which can be fragmented by 266-nm light. The selectivity offered by photodissociation allows facile identification of the peptides of interest even in complex mixtures, and subsequent selective radical directed backbone fragmentation pinpoints the site of modification. This combination greatly simplifies data analysis and provides more confident assignments.     

In-source decay characteristics of peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In-source decay (ISD) of peptides, coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, has been examined to determine the influence of the matrix, the susceptibility of amino-acid residues to ISD, and the effect of extraction delay times. Out of nine di- and tri-hydroxybenzoic acids and three cinnamic derivatives tested, the most suitable matrix for ISD was 2,5-dihydroxybenzoic acid. The amine bond at Xxx-Gly and Xxx-Val residues was less susceptible than other amino-acid residues to ISD; however, the more sensitive residue(s) were not as clear. Using a peptide that gave the y(n)- and (z(n) + 2)-series product ions, it was confirmed that amide-bond cleavage (formation of the y(n)-series ions) accompanied metastable peaks, whereas metastable peaks were never observed with amine-bond cleavage [formation of the (z(n) + 2)-series ions]. Furthermore, abundant c(n)-series ions, which originate from amine-bond cleavage on the peptide backbone, were observed whenever a minimum delay time of 38 ns or continuous extraction was used to obtain spectra. These data indicate that amine-bond cleavage in ISD takes place on the ionization time scale before the energy randomization is completed.     

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.     

PAS-cal: a Generic Recombinant Peptide Calibration Standard for Mass Spectrometry

We describe the design, preparation, and mass-spectrometric characterization of a new recombinant peptide calibration standard with uniform biophysical and ionization characteristics for mass spectrometry. “PAS-cal” is an artificial polypeptide concatamer of peptide cassettes with varying lengths, each composed of the three small, chemically stable amino acids Pro, Ala, and Ser, which are interspersed by Arg residues to allow site-specific cleavage with trypsin. PAS-cal is expressed at high yields in Escherichia coli as a Small Ubiquitin-like MOdifier (SUMO) fusion protein, which is easily purified and allows isolation of the PAS-cal moiety after SUMO protease cleavage. Upon subsequent in situ treatment with trypsin, the PAS-cal polypeptide yields a set of four defined homogeneous peptides in the range from 2 to 8 kDa with equal mass spacing. ESI-MS analysis revealed a conveniently interpretable raw spectrum, which after deconvolution resulted in a very simple pattern of four peaks with similar ionization signals. MALDI-MS analysis of a PAS-cal peptide mixture comprising both the intact polypeptide and its tryptic fragments revealed not only the four standard peptides but also the singly and doubly charged states of the intact concatamer as well as di- and trimeric adduct ion species between the peptides, thus augmenting the observable m/z range. The advantageous properties of PAS-cal are most likely a result of the strongly hydrophilic and conformationally disordered PEG-like properties of the PAS sequences. Therefore, PAS-cal offers an inexpensive and versatile recombinant peptide calibration standard for mass spectrometry in protein/peptide bioanalytics and proteomics research, the composition of which may be further adapted to fit individual needs.

Analysis of aspartyl peptide degradation products by high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry

A reversed-phase HPLC method for the analysis of degradation products of the model aspartyl tripeptides Phe-Asp-GlyNH2 and Gly-Asp-PheNH2 after incubation at pH 2 and 10 was developed. Most of the compounds could be separated with a gradient of acetonitrile in water containing 0.1% trifluoroacetic acid. Resolution of the isomeric pairs L-Phe-alpha-L-Asp-GlyNH2/L-Phe-beta-L-Asp-GlyNH2 and L-Phe-alpha-D-Asp-GlyOH/L-Phe-beta-D-Asp-GlyOH was achieved with a gradient of acetonitrile in phosphate buffer, pH 5.0. Under acidic conditions the major degradation pathway was cleavage of the peptide backbone amide bonds yielding dipeptides and amino acids, C-terminal deamidation as well as formation of succidinimyl peptides. At alkaline pH both deamidation of the C-terminal amide as well as isomerization and concomitant enantiomerization of Asp were observed. The peaks were identified both by reference substances and by online electrospray mass spectrometry. The results were compared to a previous developed capillary electrophoresis method. Diastereomeric pairs ofpeptides that could not be separated by capillary electrophoresis were resolved by HPLC while the separation of corresponding pairs of alpha- and beta-Asp peptides was not always achieved by HPLC in contrast to capillary electrophoresis illustrating that both techniques can be complimentary in peptide analysis.     

A method for rapid protease substrate evaluation and optimization

We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.     

Isotope pattern vector based tandem mass spectral data calibration for improved peptide and protein identification

Tandem mass spectra contain noisy peaks which make peak picking for peptide identification difficult. Moreover, all spectral peaks can be shifted due to systematic measurement errors. In this paper, a novel use of an isotope pattern vector (IPV) is proposed for denoising and systematic measurement error prediction. By matching the experimental IPVs with the theoretical IPVs of candidate fragment ions, true ionic peaks can be identified. Furthermore, these identified experimental IPVs and their corresponding theoretical IPVs are used in an optimization process to predict the systematic measurement error associated with the target spectrum. In return, the subsequent spectral data calibration based on the predicted systematic measurement error enhances the data quality. We show that such an integrated denoising and calibration process leads to significantly improved peptide and protein identification. Different from the commonly employed chemical calibration methods, our IPV‐based method is a purely computational method for individual spectra analysis and globally optimizes the use of spectral data. Copyright © 2009 John Wiley & Sons, Ltd.     

Enzyme-cleavable linkers for peptide and glycopeptide synthesis

Hydroxymethylphenoxy linkers that are commonly used in solid phase peptide synthesis are surprisingly susceptible to efficient cleavage by the protease chymotrypsin with a broad range of amino acid residues being tolerated at the scissile bond; this enzyme-cleavable linker system has been applied to peptide and glycopeptide synthesis.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.