The proteolytic activity of insulin‐degrading enzyme: a mass spectrometry study

The prominent role that insulin‐degrading enzyme (IDE) has on amyloidogenic peptides degradation has recently boosted a lot of attention toward this enzyme. Although many substrates are known to be degraded by IDE, little is known about the changes in the proteolytic activity of the enzyme upon modification of environmental factors. In a previous work we have already shown the great potentiality of atmospheric pressure/laser desorption ionization‐mass spectrometry (AP/MALDI‐MS) for studying the interaction between IDE and insulin. Here, the activity of IDE was investigated regarding cleavage sites' preferentiality upon modification of environmental factors by AP/MALDI‐MS. The roles that IDE/insulin concentration ratio, reaction time, adenosine 5′‐triphosphate (ATP) and metal ions (Zn and Cu) have on the insulin cleavage pattern produced by IDE are investigated and a plausible interpretation involving the proteolytic action of the different IDE oligomeric forms is proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐chip solid‐phase extraction pre‐concentration/focusing substrates coupled to atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis

Atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP‐MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP‐MALDI suffers from less‐than‐optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal‐to‐noise ratio (S/N) gains observed when an on‐chip dynamic pre‐concentration/focusing approach is coupled to AP‐MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP‐MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200–900 for hydrophobic substrates, and 150–400 for weak cation‐exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/µL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter‐day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP‐MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads. Copyright © 2009 John Wiley & Sons, Ltd.     

Immobilized endoproteinase Glu‐C to magnetic bead cellulose as a tool in proteomic analysis

Magnetic bead cellulose activated with divinyl sulfone was used for the immobilization of Staphylococcus aureus endoproteinase Glu‐C (EC 3.4.21.19). The immobilized proteinase was characterized by increased thermostability, by decreased self‐cleavage activity, and a possibility of repeated use. The prepared immobilized enzyme was applied for the proteolytic cleavage of α‐casein and BSA under different conditions (different composition of buffers, different pH, and different time of digestion). The possibilities of the direct use of enzyme reaction products for MALDI TOF MS analysis were shown.     

Analysis of human plasma lipids and soybean lecithin by means of high‐performance thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

An improved analytical strategy for the analysis of complex lipid mixtures using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) in combination with high‐performance thin‐layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and ‐sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post‐source decay (PSD) product ion analysis was performed in order to obtain further structural information (head‐ and acyl‐group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R‐250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI‐MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on‐line HPTLC/MALDI‐MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI‐MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 µL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF‐MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples. Copyright © 2009 John Wiley & Sons, Ltd.     

High‐Mass MALDI‐MS Analysis for the Investigation of Protein Encapsulation within an Engineered Capsid Forming Protein

Chemical cross‐linking combined with MALDI ‐MS was applied to structural analysis of a protein nanocontainer. Specifically, an engineered variant of lumazine synthase from Aquifex aeolicus (AaLS ‐13) was investigated that self‐assembles into a capsid‐like structure and is known to encapsulate other proteins by Coulombic attraction. Two complementary soft ionization techniques, MALDI ‐MS and native ESI ‐MS , were utilized to map the subunit stoichiometry of the high molecular weight capsid. In accordance with the previously reported cryo‐electron microscopy structure of this protein container, only pentameric subunits were detected. This study highlights the possibility to map subunit stoichiometry via chemical cross‐linking with glutaraldehyde followed by MALDI ‐MS . The same approach was used to study protein‐protein interactions during encapsulation of GFP (+36) by the AaLS ‐13 capsid. Heterocomplexes between GFP (+36) and AaLS ‐13 multimers were not observed when mixed at maximal loading capacity (AalS‐13 monomer:GFP (+36) 4:1). This is in agreement with the known fast encapsulation of GFP (+36) by the protein capsid, which essentially removes any free GFP (+36) from the solution. Exceeding the maximal loading capacity by addition of excess GFP (+36) results in aggregation.     

Direct identification of trypanosomatids by matrix‐assisted laser desorption ionization–time of flight mass spectrometry (DIT MALDI‐TOF MS)

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix‐assisted laser desorption ionization/time of flight mass spectrometry (MALDI‐TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI‐TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI‐TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI‐TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software‐assisted identification at the strain level. Overall, this study shows the importance of MALDI‐TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry‐based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.     

A novel synthetic strategy for copolymers of vinyl alcohol: Radical copolymerization of alkoxyvinylsilanes with styrene and oxidative transformation of C?Si(OR)2Me into C?OH in the copolymers to afford poly(vinyl alcohol‐ran‐styrene)s

As a novel synthetic strategy for copolymers of vinyl alcohol, we propose herein copolymerization of alkoxyvinylsilanes with other vinyl monomers, followed by oxidative cleavage of the alkoxysilyl groups attached to the main chain of the resulting copolymers. Radical copolymerization of di(isobutoxy)methylvinylsilane 1 with styrene afforded poly( 1 ‐ran‐styrene)s with a variety of compositions of both repeating units, although the M_n's (<9000) and yields (<35%) were rather low. The oxidative cleavage of the alkoxysilyl groups in the copolymers with m‐chloroperbenzoic acid proceeded efficiently, giving poly(vinyl alcohol‐ran‐styrene)s, which were soluble in common organic solvents. The structures of the poly(vinyl alcohol‐ran‐styrene)s were characterized by NMR, GPC, elemental analysis, and matrix‐assisted laser desorption time‐of‐flight mass spectrometry (MALDI‐TOF‐MS). © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 3648–3658, 2007     

The use of matrix coating assisted by an electric field (MCAEF) to enhance mass spectrometric imaging of human prostate cancer biomarkers

In this work, we combined a newly developed matrix coating technique – matrix coating assisted by an electric field (MCAEF) and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to enhance the imaging of peptides and proteins in tissue specimens of human prostate cancer. MCAEF increased the signal‐to‐noise ratios of the detected proteins by a factor of 2 to 5, and 232 signals were detected within the m/z 3500–37500 mass range on a time‐of‐flight mass spectrometer and with the sinapinic acid MALDI matrix. Among these species, three proteins (S100‐A9, S100‐A10, and S100‐A12) were only observed in the cancerous cell region and 14 proteins, including a fragment of mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase kinase 2, a fragment of cAMP‐regulated phosphoprotein 19, 3 apolipoproteins (C‐I, A‐I, and A‐II), 2 S100 proteins (A6 and A8), β‐microseminoprotein, tumor protein D52, α‐1‐acid glycoprotein 1, heat shock protein β‐1, prostate‐specific antigen, and 2 unidentified large peptides at m/z 5002.2 and 6704.2, showed significantly differential distributions at the p < 0.05 (t‐test) level between the cancerous and the noncancerous regions of the tissue. Among these 17 species, the distributions of apolipoprotein C‐I, S100‐A6, and S100‐A8 were verified by immunohistological staining. In summary, this study resulted in the imaging of the largest group of proteins in prostate cancer tissues by MALDI‐MS reported thus far, and is the first to show a correlation between S100 proteins and prostate cancer in a MS imaging study. The successful imaging of the three proteins only found in the cancerous tissues, as well as those showing differential expressions demonstrated the potential of MCAEF‐MALDI/MS for the in situ detection of potential cancer biomarkers. Copyright © 2015 John Wiley & Sons, Ltd.     

Extending matrix‐assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates

Mass spectrometry (MS)‐based high‐throughput screening (HTS) has tremendous potential as an alternative to current screening methods due to its speed, sensitivity, reproducibility and label‐free readout. We recently reported that a new generation matrix‐assisted laser desorption/ionization triple quadrupole (MALDI‐QqQ) mass spectrometer is ideally suited for a variety of enzyme assays and screening protocols. However, all the targets measured to date had peptide substrates that were easily monitored by selected ion monitoring (SIM) without interference from the MALDI matrix. To further extend the application to enzymes with small molecule, non‐peptide substrates, we evaluated this method for measuring enzyme activity and inhibition of acetylcholinesterase (AChE). Due to the potential of MALDI matrix interference, multiple reaction monitoring (MRM) was investigated for selective MS/MS transitions and to accurately measure the conversion of acetylcholine into choline. Importantly, ionization, detection and MRM transition efficiency differences between the substrate and product can be overcome by pre‐balancing the MRM transitions during method development, thus allowing for a direct readout of the enzyme activity using the ratio of the substrate and product signals. Further validation of the assay showed accurate concentration‐dependent inhibition measurements of AChE with several known inhibitors. Finally, a small library of 1008 drug‐like compounds was screened at a single dose (10 µM) and the top 10 inhibitors from this primary screen were validated in a secondary screen to determine the rank order of inhibitory potency for each compound. Collectively, these data demonstrate that a MALDI‐QqQMS‐based readout platform is amenable to measuring small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs. Copyright © 2009 John Wiley & Sons, Ltd.     

Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

MALDI‐based intact spore mass spectrometry of downy and powdery mildews

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI‐TOF MS‐based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer‐based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10⁹ spores per ml. The best peptide/protein profiles (in terms of signal‐to‐noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of l‐tryptophan and l‐kynurenine derivatized with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole by LC‐MS/MS on a triazole‐bonded column and their quantification in human serum

The concentrations of l ‐tryptophan (Trp) and the metabolite l ‐kynurenine (KYN) can be used to evaluate the in‐vivo activity of indoleamine 2,3‐dioxygenase (IDO) and tryptophan 2,3‐dioxygenase (TDO). As such, a novel method involving derivatization of l ‐Trp and l ‐KYN with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS) and separation by high‐performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection on a triazole‐bonded column (Cosmosil HILIC®) was developed to determine their concentrations. The optimized mobile phase, CH₃CN/10 mm ammonium formate in H₂O (pH 5.0) (90:10, v/v) eluted isocratically, resulted in satisfactory separation and MS/MS detection of the analytes. The detection limits of l ‐Trp and l ‐KYN were approximately 50 and 4.0 pm , respectively. The column temperature affected the retention behaviour of the Trp and KYN derivatives, with increased column temperatures leading to increased capacity factors; positive enthalpy changes were revealed by van't Hoff plot analyses. Using the proposed LC‐MS/MS method, l ‐Trp and l ‐KYN were successfully determined in 10 μL human serum using 1‐methyl‐l ‐Trp as an internal standard. The precision and recovery of l ‐Trp were in the ranges 2.85–9.29 and 95.8–113%, respectively, while those of l ‐KYN were 2.51–16.0 and 80.8–98.2%, respectively. The proposed LC‐MS/MS method will be useful for evaluating the in vivo activity of IDO or TDO. Copyright © 2016 John Wiley & Sons, Ltd.     

Syntheses of 2‐Keto‐3‐deoxy‐D‐xylonate and 2‐Keto‐3‐deoxy‐L‐arabinonate as Stereochemical Probes for Demonstrating the Metabolic Promiscuity of Sulfolobus solfataricus Towards D‐Xylose and L‐Arabinose

Practical syntheses of 2‐keto‐3‐deoxy‐D ‐xylonate (D ‐KDX) and 2‐keto‐3‐deoxy‐L ‐arabinonate (L ‐KDA) that rely on reaction of the anion of ethyl 2‐[(tert‐butyldimethylsilyl)oxy]‐2‐(dimethoxy phosphoryl) acetate with enantiopure glyceraldehyde acetonide, followed by global deprotection of the resultant O‐silyl‐enol esters, have been developed. This has enabled us to confirm that a 2‐keto‐3‐deoxy‐D ‐gluconate aldolase from the archaeon Sulfolobus solfataricus demonstrates good activity for catalysis of the retro‐aldol cleavage of both these enantiomers to afford pyruvate and glycolaldehyde. The stereochemical promiscuity of this aldolase towards these enantiomeric aldol substrates confirms that this organism employs a metabolically promiscuous pathway to catabolise the C5‐sugars D ‐xylose and L ‐arabinose.     

Disperse red 1 end capped oligoesters. Synthesis by noncatalyzed ring opening oligomerization and structural characterization

The synthesis of novel azofunctional oligoesters through bulk ring opening of ε‐caprolactone and D ,L ‐lactide (LA) at 100 and 130 °C, respectively, mediated by N‐ethyl‐N‐(2‐hydroxyethyl)‐4‐(4‐nitrophenylazo)aniline (Disperse Red 1) (DR1) is described. The synthetic procedure allows “clean” products because no catalysts were used in the reaction. Moreover, DR1 moiety is showed for the first time to promote the ring opening of cyclic esters. The molecular structure of the obtained oligoesters was established by NMR spectroscopy, MALDI ToF MS and electrospray ionization mass spectrometry (ESI MS). ESI‐MS/MS fragmentation experiments were used to demonstrate the nature of the chain end groups (hydroxyl and DR1). Intermolecular transesterification reactions were proved by mass spectrometry studies at least in the case of LA oligomerization. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 534–547, 2009     

Rapid Detection of Irreversible Acetylcholineasterase Inhibitor by Mass Spectrometry Assay

Here we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix‐assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI‐FTMS) for screening irreversible AChE inhibitors. Due to its good salt‐tolerance and low sample consumption, MALDI‐FTMS could facilitate rapid detection, especially detection in real application. AChE activity was determined through calculating abundance of substrate and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) concentrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibitions still kept good correlation with concentration of OPs. Finally, this AChE‐inhibited method was applied to screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of decedents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following detecting in GC‐MS/MS, the possible death reason of these three decedents was confirmed, and another decedent actually died for sumicidin, a non‐AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by detecting enzyme activity in MALDI‐FTMS provided fast and accurate analysis results and excluded another toxicants not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.     

Matrix‐assisted laser desorption/ionization tissue profiling of secretoneurin in the nucleus accumbens shell from cocaine‐sensitized rats

Proteins in the nucleus accumbens mediate many cocaine‐induced behaviors. In an effort to measure changes in nucleus accumbens protein expression as potential biomarkers for addiction, coronal tissue sections were obtained from rats that developed behavioral sensitization after daily administration of cocaine, or from daily saline‐treated controls. The tissue sections were subjected to matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS) profiling and tissue imaging. For profiling experiments, brain sections were manually spotted with matrix over the nucleus accumbens, a brain region known to regulate cocaine sensitization. Summed mass spectra (10 000 laser shots, grid) were acquired and spectra were aligned to reference peaks. Using bioinformatics tools, eight spectral features were found to be altered by cocaine treatment. Based on additional sequencing experiments with MALDI tandem MS and database searches of measured masses, secretoneurin (m/z 3653) was identified as having an increased expression. In addition, the distribution of m/z 3653 in the nucleus accumbens was determined by MALDI tissue imaging, and the increased expression of its precursor protein, secretogranin II, was verified by immunoblotting. Copyright © 2009 John Wiley & Sons, Ltd.     

Block length determination of the block copolymer mPEG‐b‐PS using MALDI‐TOF MS/MS

The molar mass determination of block copolymers, in particular amphiphilic block copolymers, has been challenging with chromatographic techniques. Therefore, methoxy poly(ethylene glycol)‐b‐poly(styrene) (mPEG‐b‐PS) was synthesized by atom transfer radical polymerization (ATRP) and characterized in detail not only by conventional chromatographic techniques, such as size exclusion chromatography (SEC), but also by matrix‐assisted laser/desorption ionization tandem mass spectrometry (MALDI‐TOF MS/MS). As expected, different molar mass values were obtained in the SEC measurements depending on the calibration standards (either PEG or PS). In contrast, MALDI‐TOF MS/MS analysis allowed the molar mass determination of each block, by the scission of the weakest point between the PEG and PS block. Thus, fragments of the individual blocks could be obtained. The PEG block showed a depolymerization reaction, while for the PS block fragments were obtained in the monomeric, dimeric, and trimeric regions as a result of multiple chain scissions. The block length of PEG and PS could be calculated from the fragments recorded in the MALDI‐TOF MS/MS spectrum. Furthermore, the assignment of the substructures of the individual blocks acquired by MALDI‐TOF MS/MS was accomplished with the help of the fragments that were obtained from the corresponding homopolymers. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Mass spectrometry of peptides and proteins using digestion by a grape cysteine protease at pH 3

Cysteine protease from grapevine (Vitis vinifera) belongs to those resistant proteins, which survive the process of vinification and can therefore be detected as wine components. Its amino acid sequence shows a homology to other members of the papain family, but the enzyme has only partially been explored so far. In order to get more biochemical information with the help of mass spectrometry (MS), wine proteins were collected by ultrafiltration and separated by gel permeation chromatography. The purified enzyme surprisingly displayed a high molecular mass value of around 200 kDa, indicating a possible oligomeric status and aggregation, as it entered only negligibly the separating 10% gel during polyacrylamide gel electrophoresis. The isoelectric point (pI) value of 3.6 was determined by chromatofocusing. Matrix‐assisted laser desorption/ionization (MALDI)‐MS was employed to evaluate the cleavage specificity and usefulness of the isolated cysteine protease in protein and peptide research. A potential applicability could be anticipated from the efficient digestion performance in volatile ammonium formate buffers at pH 3. Common peptides were digested and the resulting products analyzed by MS/MS sequencing. Then, mixtures of protein standards and extracted barley nuclear proteins were processed in the same way. Grape cysteine protease is nonspecific but shows a certain preference for Arg, Lys, and also Leu residues. Compared with papain, it seems not to require fully the presence of a large hydrophobic residue adjacent to that at the cleavage site. The enzyme is suitable for protein research as it produces peptides of a reasonable length in acidic pH.     

Expression,Purification and Characterization of a Bifunctional α‐L‐Arabinofuranosidase/β‐D‐Xylosidase from Trichoderma Koningii G‐39

A gene of α‐L ‐arabinofuranosidase (Abf) from Trichoderma koningii G‐39 was successfully expressed in Pichia pastoris. The recombinant enzyme was purified to > 90% homogeneity by a cation‐exchanged chromatography. The purified enzyme exhibits both α‐L ‐arabinofuranosidase and β‐D ‐xylosidase (Xyl) activities with p‐nitrophenyl‐α‐L ‐arabionfuranoside (pNPAF) and 2,4‐dinitrophenyl‐β‐D ‐xylopyanoside (2,4‐DNPX) as substrate, respectively. The stability and the catalytic feature of the bifunctional enzyme were characterized. The enzyme was stable for at least 2 h at pH values between 2 and 8.3 at room temperature when assayed for Abf and Xyl activities. Enzyme activity decreased dramatically when the pH exceeded 9.5 or dropped below 1.5. The enzyme lost 35% of Abf activity after incubation at 55 °C for 2 h, but retained 95% of Xyl activity, with 2,4‐DNXP as substrate, under the same conditions. Further investigation of the active site topology of both enzymatic functions was performed with the inhibition study of enzyme activities. The results revealed that methyl‐α‐L ‐arabinofuranoside inhibition is noncompetitive towards 2,4‐DNPX as substrate but competitive towards pNPAF. Based on the thermal stability and the inhibition studies, we suggest that the enzymatic reactions of Abf and Xyl are performed at distinct catalytic sites. The recombinant enzyme possesses both the retaining transarabinofuranosyl and transxylopyranosyl activities, indicating both enzymatic reactions proceed through a two‐step, double displacement mechanism.     

The photobase generator nifedipine as a novel matrix for the detection of polyphenols in matrix‐assisted laser desorption/ionization mass spectrometry

Matrix assisted laser desorption/ionization mass spectrometry (MALDI‐MS) is widely used for the detection and analysis of ionizable compounds. However, the method has less potential for the analysis of neutral compounds, such as polyphenols, owing to their lack of favorable proton‐attachment or ‐removal groups. In this study, we reported for the first time that nifedipine (2,6‐dimethyl‐3,5‐dicarbomethoxy‐4‐(2‐nitrophenyl)‐1,4‐dihydropyridine), which is a strong photobase generator commonly used in polymerization, can abstract protons from neutral compounds in negative mode‐MALDI experiments. When nifedipine (5 mg/ml) was used as a matrix reagent, the limit of detection (LOD) for epigallocatechin‐3‐O‐gallate (EGCG) was determined to be 100 fmol/spot, which constitutes >50‐fold improvement compared to the LOD obtained when trans‐3‐indoleacrylic acid, a matrix reagent previously reported for polyphenol detection, was used. Of the dihydropyridines investigated, only nifedipine facilitated the detection of EGCG, suggesting that the nitrosophenyl pyridine derivative of nifedipine formed by photoreduction under laser irradiation at 355 nm plays a crucial role in detecting polyphenols in negative mode. Reduced MS detection of 5‐O‐methylnaringenin indicated that nifedipine may preferably remove a proton from the 5‐position OH group in the A ring of the flavonoid skeleton. The significant MS detection by nifedipine was extensively observed for polyphenols including flavones, flavonones, chalcones, stilbenoids and phenolic acids. In conclusion, nifedipine can act as a novel matrix for improving polyphenol detection by MALDI‐MS in negative mode. Copyright © 2016 John Wiley & Sons, Ltd.