Detection of non-covalent protein interactions by 'intensity fading' MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors

Among the main objectives of biomedical and proteomic research is to identify non-covalent interactions involving proteins. Here we provide a detailed protocol to apply matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry for such a purpose using proteases and protease inhibitors in complex biological samples. Our methodology is based on monitoring the reduction in intensity of inhibitors' mass spectrometric signals when their protease target is added to the MALDI sample. The versatility of the protocol permits the target to be added in a soluble form (direct protocol) or immobilized form (indirect protocol). The 'intensity fading' phenomenon is greatly favored when the binding assay is carried out in the sub-micromolar range and the interacting partners occur in mixtures of non-binding compounds. This protocol can be completed in 10 h, taking 20 or 30 min per sample to perform the mass spectrometric data acquisition, depending on whether a soluble or an immobilized target is used.     

蛋白酶及其抑制剂关键活性位点研究进展

蛋白酶广泛存在于生物体中,参与分解蛋白质,维持生物体正常的生命活动.蛋白酶抑制剂通过与蛋白酶活性位点结合调控靶蛋白酶活性,从而影响蛋白质代谢.蛋白酶及其抑制剂关键氨基酸的突变,可以影响其生理功能、稳定性、催化活性、抑制特异性等.通过挖掘自然界蛋白酶及其抑制剂的各种突变体,分析它们的关键活性位点,并运用蛋白质工程手段改造...     

Alterations in proteases,protease inhibitors and ecdysone levels: a profile of stress in insects

• 1.1. A comparison of proteolytic and protease inhibitory activity, and ecdysteroid levels in body fluids was made between normal larvae of the flesh fly, Sarcophaga bullata, and those that had been water-stressed for two days.
• 2.2. The course of proteolytic activity in water stressed flies decreases 6 hr after beginning the experiment and remains low in comparison with control flies.
• 3.3. The course of protease inhibitors exhibits a mirror image pattern to proteases.
• 4.4. Ecdysteroid pattern shows two peaks in control animals: minor at 24 hr and major at pupariation, in experimental animals: at 1 hr, at 6 hr and at white pupal stage.

     

Interaction of serine protease inhibitors and substrates with human uterine estrogen receptor

The human uterine estrogen receptor has a site which regulates estrogen binding and which structurally resembles the substrate binding site of chymotrypsin. The hormone binding capacity and the affinity of the receptor is decreased in the presence of 4 mM serine protease inhibitors tosyl-lysine chloromethyl ketone and diisopropylfluorophosphate and the protease substrates tryptophan methyl ester and toluene sulphonyl-arginine methyl ester. The protease inhibitors tosylamide-phenylethyl-chloromethyl ketone and phenyl methyl sulphonyl fluoride caused an increase in the binding capacity whereas the affinity was decreased.     

Peptide substrates and inhibitors of the HIV-1 protease

Oligopeptides containing the consensus retroviral protease cleavage sequence Ser/Thr-X-Y-Tyr/Phe-Pro are substrates for purified recombinant HIV-1 protease with Km's in the millimolar range. The minimum sequence containing the consensus pentapeptide which serves as a good substrate is a heptapeptide spanning the P4-P3' residues. Substitution of reduced Phe-Pro or Tyr-Pro dipeptide isosteres or the statine analog 3-hydroxy-4-amino-5-phenylpentanoic acid for the scissile dipeptide afforded inhibitors of HIV-1 protease with Ki values in the micromolar range, three orders of magnitude better in affinity than the corresponding substrates. Inhibitors of HIV-1 protease may provide a novel and potentially useful therapeutic approach to the treatment of acquired immune deficiency syndrome (AIDS).     

Down-regulation of human extracellular cysteine protease inhibitors by the secreted staphylococcal cysteine proteases, staphopain A and B

Of seven human cystatins investigated, none inhibited the cysteine proteases staphopain A and B secreted by the human pathogen Staphylococcus aureus. Rather, the extracellular cystatins C, D and E/M were hydrolyzed by both staphopains. Based on MALDI-TOF time-course experiments, staphopain A cleavage of cystatin C and D should be physiologically relevant and occur upon S. aureus infection. Staphopain A hydrolyzed the Gly11 bond of cystatin C and the Ala10 bond of cystatin D with similar Km values of approximately 33 and 32 microM, respectively. Such N-terminal truncation of cystatin C caused >300-fold lower inhibition of papain, cathepsin B, L and K, whereas the cathepsin H activity was compromised by a factor of ca. 10. Similarly, truncation of cystatin D caused alleviated inhibition of all endogenous target enzymes investigated. The normal activity of the cystatins is thus down-regulated, indicating that the bacterial enzymes can cause disturbance of the host protease-inhibitor balance. To illustrate the in vivo consequences, a mixed cystatin C assay showed release of cathepsin B activity in the presence of staphopain A. Results presented for the specificity of staphopains when interacting with cystatins as natural protein substrates could aid in the development of therapeutic agents directed toward these proteolytic virulence factors.     

Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease

Retroviruses code for a virus-specific protease which is essential for polyprotein processing and viral infectivity. The human immune deficiency virus-1 protease is an aspartic protease of 9 kDa which was synthesized by recombinant DNA technology and arises by autocatalytic processing from a polyprotein precursor which has recently been demonstrated by use of a protease-specific monoclonal antibody. The protease was shown to form dimers. Here we demonstrate that synthetic peptides can be used as both model substrates as well as inhibitors for investigation of the protease. 14 synthetic peptides, 7-18 amino acids in length, containing putative protease cleavage sites of the viral polyprotein gag and pol precursors, have been analyzed with the partially purified protease by the use of high performance liquid chromatography. In seven cases, where cleavage was observed, the length of the peptides did not significantly influence the cleavage efficiencies, heptapeptides being large enough as model substrates. No cleavage was observed with a protein preparation purified in parallel from control bacteria not expressing the human immune deficiency virus-1 protease. The protease was not only able to cut next to a proline but also between other peptides indicating that the proline is not a prerequisite. Three peptides with either reduced bonds at the cleavage site or a substitution by statin were inhibitory while another uncleaved substrate was not. The usefulness of small model substrates for characterization of the protease is further demonstrated by determination of a kinetic optimum pH (3.5-5.5) and incubation temperature (37 degrees C).     

Influence of extracellular proteins,proteases and protease inhibitors on grapevine somatic embryogenesis

An embryogenic grapevine rootstock cell suspension, continuously grown in the presence of auxin, was predominantly composed of proembryogenic masses. When transferred to an auxin-free medium, grapevine somatic embryos developed but were rapidly blocked at the heart stage. This inhibition has been related to the presence of extracellular macromolecules (Coutos-Thévenot et al., 1992a). In this study, the initial cell population density has been found to influence markedly embryo development. Inoculations below 5·10³ cells per ml were required to obtain fully grown cotyledonary embryos. Interestingly, extracellular proteins of molecular weights of 32, 34, 48 and 52 kDa accumulated in cultures grown at high population cell densities and disappeared in cultures inoculated at densities below 5·10³ cells per ml. Protein fractions partially purified by ion exchange chromatography caused both an early inhibition of embryogenesis and a stimulation of secondary embryogenesis. Moreover, to test for the possibility of modulating embryo development through alterations of extracellular proteins, cultures were supplemented with proteases and protease inhibitors. The addition of trypsin increased the rate of embryo development only in cultures inoculated at a low cell population density. Conversely, the protease inhibitor aprotinin inhibited development, arresting embryos at globular and heart stages. Together, these results provide evidence that extracellular proteins modulate somatic embryogenesis and suggest that an extracellular proteolitic mechanism could be implicated in development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Design, synthesis, and evaluation of azapeptides as substrates and inhibitors for human rhinovirus 3C protease

A series of azapeptides was prepared and assessed as inhibitors of the human rhinovirus 3C protease. Boc-VLFaQ-OPh was a slow-turnover substrate that gave transient (ca. 1-2 h) inhibition as it underwent hydrolysis. Boc-VLFaG-OPh gave very slow but essentially irreversible inhibition.     

Annotation of the human cerebrospinal fluid lipidome using high resolution mass spectrometry and a dedicated data processing workflow

Introduction

Due to its proximity with the brain, cerebrospinal fluid (CSF) could be a medium of choice for the discovery of biomarkers of neurological and psychiatric diseases using untargeted analytical approaches.

Objectives

This study explored the CSF lipidome in order to generate a robust mass spectral database using an untargeted lipidomic approach.

Methods

Cerebrospinal fluid samples from 45 individuals were analyzed by liquid chromatography coupled to high-resolution mass spectrometry method (LC-HRMS). A dedicated data processing workflow was implemented using XCMS software and adapted filters to select reliable features. In addition, an automatic annotation using an in silico lipid database and several MS/MS experiments were performed to identify CSF lipid species.

Results

Using this complete workflow, 771 analytically relevant monoisotopic lipid species corresponding to 550 unique lipids which represent five major lipid families (i.e., free fatty acids, sphingolipids, glycerophospholipids, glycerolipids, and sterol lipids) were detected and annotated. In addition, MS/MS experiments enabled to improve the annotation of 304 lipid species. Thanks to LC-HRMS, it was possible to discriminate between isobaric and also isomeric lipid species; and interestingly, our study showed that isobaric ions represent about 50 % of the total annotated lipid species in the human CSF.

Conclusion

This work provides an extensive LC/HRMS database of the human CSF lipidome which constitutes a relevant foundation for future studies aimed at finding biomarkers of neurological disorders.

     

Simultaneous determination of HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in human plasma by high-performance liquid chromatography-tandem mass spectrometry

We report a precise and accurate method for simultaneous quantification of protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in plasma. An internal standard was added to samples prior to protein precipitation with acetonitrile followed by addition of ammonium formate buffer. Analysis was by HPLC-MS/MS. Calibration curves were validated over concentration ranges encompassing both subtherapeutic and potentially 'toxic' drug concentrations. Inter- and intra-assay variation were below 11% and PI recovery was above 87%. The bioanalytical method described is successfully applied to measure PI concentrations obtained from clinical pharmacokinetic studies and routine therapeutic drug monitoring (TDM).     

Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.     

One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry

We describe a simple and direct zymographic method for the detection of proteases using quenched fluorescent substrates. The proteases were separated using one- and two-dimensional electrophoresis, and the gel subsequently was incubated with the quenched fluorescent substrate. After a short incubation, the released fluorescence allowed the localization of the proteases directly using UV light. The protease spots could then be cut directly from the gel and processed for identification by mass spectrometry. This method could easily be used to develop or test whether a substrate is specific or not and also to detect the proteases that are able to cleave this substrate in a complex biological fluid. This also allowed direct identification of proteases without complex purification.     

Determination of amprenavir, a HIV-1 protease inhibitor, in human seminal plasma using high-performance liquid chromatography-tandem mass spectrometry

A HPLC-MS-MS method to measure amprenavir in human seminal plasma has been developed and validated. The procedure uses stable, isotopically labeled 13C6-amprenavir as an internal standard and 100 microl of sample. The method is accurate (bias less than or equal to 7.2%) and precise (within- and between-day RSDs less than or equal to 4.2%) over the dynamic range of 30-4,000 ng/ml. Recently, this simple and sensitive method was used to determine amprenavir concentrations in seminal samples collected from HIV-1 positive subjects receiving amprenavir antiretroviral therapy as part of a multicenter clinical trial.     

Class prediction and discovery using gene microarray and proteomics mass spectroscopy data: curses,caveats, cautions

MOTIVATION: Two practical realities constrain the analysis of microarray data, mass spectra from proteomics, and biomedical infrared or magnetic resonance spectra. One is the 'curse of dimensionality': the number of features characterizing these data is in the thousands or tens of thousands. The other is the 'curse of dataset sparsity': the number of samples is limited. The consequences of these two curses are far-reaching when such data are used to classify the presence or absence of disease. RESULTS: Using very simple classifiers, we show for several publicly available microarray and proteomics datasets how these curses influence classification outcomes. In particular, even if the sample per feature ratio is increased to the recommended 5-10 by feature extraction/reduction methods, dataset sparsity can render any classification result statistically suspect. In addition, several 'optimal' feature sets are typically identifiable for sparse datasets, all producing perfect classification results, both for the training and independent validation sets. This non-uniqueness leads to interpretational difficulties and casts doubt on the biological relevance of any of these 'optimal' feature sets. We suggest an approach to assess the relative quality of apparently equally good classifiers.     

Protease inhibitors and human plasmin: interaction in a whole plasma system.

Using trace amounts of [¹²⁵I]-plasminogen and conventional biochemical techniques, the distribution of the labeled zymogen amongst the various protease inhibitors was studied in whole plasma before and after activation with urokinase and streptokinase. A small percentage of the labeled enzyme was bound to α₂-macroglobulin while a majority was complexed to a component in plasma immunologically distinct from the well known human antiplasmins. The inhibitor was identified as α₂-antiplasmin and confirmed the existence of this antiprotease recently described by others. These data also suggest that the other antiplasmins may play a minor, yet important role in the regulation of plasmin activity under different physiological conditions.     

A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors

Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography–mass spectrometry (HPLC–MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE).     

Ultra-potent P1 modified arylsulfonamide HIV protease inhibitors: the discovery of GW0385

A novel series of P1 modified HIV protease inhibitors was synthesized and evaluated for in vitro antiviral activity against wild-type virus and protease inhibitor-resistant viruses. Optimization of the P1 moiety resulted in compounds with femtomolar enzyme activities and cellular antiviral activities in the low nanomolar range culminating in the identification of clinical candidate GW0385.     

Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

Background  

The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis.