Processing of the 3C/D Region of the Deformed Wing Virus (DWV).

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1' G2119 (KPQ/GST) as well as P1 Q2393 and P1' S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A.

Analysis of Arginylated Peptides by Subtractive Edman Degradation.

During the early studies of N-terminal arginylation, Edman degradation was widely used to identify N-terminally added Arg on protein substrates. This old method is reliable, but highly depends on the purity and abundance of samples and can become misleading unless a highly purified highly arginylated protein can be obtained. Here, we report a mass spectrometry-based method that utilizes Edman degradation chemistry to identify arginylation in more complex and less abundant protein samples. This method can also apply to the analysis of other posttranslational modifications.

Simultaneous quantification of eight hemoglobin adducts of genotoxic substances by isotope-dilution UHPLC-MS/MS.

Various genotoxic carcinogens ubiquitously present in the human environment or respective reactive metabolites form adducts in DNA and proteins, which can be used as biomarkers of internal exposure. For example, the mass spectrometric determination of Val adducts at the N-termini of hemoglobin (Hb) peptide chains after cleavage by an Edman degradation has a long tradition in occupational medicine. We developed a novel isotope-dilution UHPLC-MS/MS method for the simultaneous quantification of Val adducts of eight genotoxic substances in Hb after cleavage with fluorescein-5-isothiocyanate (FIRE procedure™). The following adducts were included [sources in square brackets]: N-(2,3-dihydroxypropyl)-Val [glycidol], N-(2-carbamoylethyl)-Val [acrylamide], N-(2-carbamoyl-2-hydroxyethyl)-Val [glycidamide], N-((furan-2-yl)methyl)-Val [furfuryl alcohol], N-(trans-isoestragole-3'-yl)-Val [estragole/anethole], N-(3-ketopentyl)-Val [1-penten-3-one], N-(3-ketooctanyl)-Val [1-octene-3-one], and N-benzyl-Val [benzyl chloride], each of which was quantified with a specific isotope-labeled standard. The limits of quantification were between 0.014 and 3.6 pmol/g Hb (using 35 mg Hb per analysis); other validation parameters were satisfactory according to guidelines of the U.S. Food and Drug Administration. The quantification in erythrocyte samples of human adults (proof of principle) showed that the median levels of Hb adducts of acrylamide, glycidamide, and glycidol were found to be significantly lower in six non-smokers (25.9, 12.2, and 4.7 pmol/g Hb, respectively) compared to those of six smokers (69.0, 44.2, and 8.6 pmol/g Hb, respectively). In summary, the method surpasses former techniques of Hb adduct quantification due to its simplicity, sensitivity, and accuracy. It can be extended continuously with other Hb adducts and will be used in epidemiological studies on internal exposure to carcinogens.

Cleavage sites and non-enzymatic self-degradation mechanism of ready-to-eat sea cucumber during storage.

The non-enzymatic degradation of ready-to-eat sea cucumber (RSC) was closely related to the quality of sea cucumber products. When stored at 37 °C for 0-30 d, the hardness of RSC decreased by 86.7% and the proportion of free water increased by 12.71%. The content of free hydroxyproline increased from 8.33 µg/g to 24.12 µg/g. Label-free quantitative proteomics analysis showed that protein was prone to break at the sites of G, Q, N, D, and L, and the peptide bonds in QI, DL, NL, RI, EF and SY were much more liable to break. Edman degradation method showed that the breakage sites of RSC were at S, D, H, E, and V. NL, NA and NG calculated by B3LYP/6-31G(d) showed that the relative free energies in the initial cyclization step were 53.20, 143.53 and 78.10 kcal/mol, respectively, which may be the rate-determining step for peptide bond cleavage.

Bioevaluation of Pheretima vulgaris Antithrombotic Extract, PvQ, and Isolation, Identification of Six Novel PvQ-Derived Fibrinolytic Proteases.

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.

Bioactivation of estragole and anethole leads to common adducts in DNA and hemoglobin.

Estragole and anethole are secondary metabolites occurring in a variety of commonly used herbs like fennel, basil, and anise. Estragole is genotoxic and carcinogenic in rodents, which depends on the formation of 1'-sulfoxyestragole after hydroxylation and subsequent sulfoconjugation catalyzed by CYP and SULT, respectively. It was hypothesized recently that anethole may be bioactivated via the same metabolic pathways. Incubating estragole with hepatic S9-fractions from rats and humans, specific adducts with hemoglobin (N-(isoestragole-3-yl)-valine, IES-Val) and DNA (isoestragole-2'-deoxyguanosine and isoestragole-2'-deoxyadenosine) were formed. An isotope-dilution technique was developed for the quantification of IES-Val after cleavage with fluorescein isothiocyanate (FITC) according to a modified Edman degradation. The same adducts, albeit at lower levels, were also detected in reactions with anethole, indicating the formation of 3'-hydroxyanethole and the reactive 3'-sulfoxyanethole. Finally, we conducted a pilot investigation in which IES-Val levels in human blood were determined during and after the consumption of an estragole- and anethole-rich fennel tea for four weeks. A significant increase of IES-Val levels was observed during the consumption phase and followed by a continuous decrease during the washout period. IES-Val may be used to monitor the internal exposure to the common reactive genotoxic metabolites of estragole and anethole, 1'-sulfoxyestragole and 3'-sulfoxyanethole, respectively.

Identification of Novel Proteins in Foam Nests of the Japanese Forest Green Tree Frog, Rhacophorus arboreus.

Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.

Sensor Array Based Determination of Edman Degradated Amino Acids Using Poly(p-phenyleneethynylene)s.

A cross-reactive optical sensor array based on poly(p-phenyleneethynylene)s (PPEs) determines Edman degraded amino acids. We report a sensor array composed of three anionic PPEs P1-P3, and their electrostatic complexes with metal ions (Fe2+ , Cu2+ , Co2+ ). We recorded distinct fluorescence intensity response patterns as "fingerprints" of this chemical tongue toward standard phenylthiohydantoin (PTH) amino acids-degradation products of the Edman process. These "fingerprints" were converted into canonical scores by linear discrimination analysis (LDA), which differentiates all of the PTH-amino acids. This array discriminates PTH-amino acid residues degraded from an oligopeptide through Edman sequencing. This approach is complementary to chromatography approaches which rely on mass spectrometry; our array offers the advantage of simplicity.

The hemoglobin adduct N-(2,3-dihydroxypropyl)-valine as biomarker of dietary exposure to glycidyl esters: a controlled exposure study in humans.

Fatty acid esters of glycidol (glycidyl esters) are heat-induced food contaminants predominantly formed during industrial deodorization of vegetable oils and fats. After consumption, the esters are digested in the gastrointestinal tract, leading to a systemic exposure to the reactive epoxide glycidol. The compound is carcinogenic, genotoxic and teratogenic in rodents, and rated as probably carcinogenic to humans (IARC group 2A). Assessment of exposure from occurrence and consumption data is difficult, as lots of different foods containing refined oils and fats may contribute to human exposure. Therefore, assessment of the internal exposure using the hemoglobin adduct of glycidol, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), may be promising, but a proof-of-principle study is needed to interpret adduct levels with respect to the underlying external exposure. A controlled exposure study was conducted with 11 healthy participants consuming a daily portion of about 36 g commercially available palm fat with a relatively high content of ester-bound glycidol (8.7 mg glycidol/kg) over 4 weeks (total amount 1 kg fat, individual doses between 2.7 and 5.2 µg/kg body weight per day). Frequent blood sampling was performed to monitor the 2,3-diHOPr-Val adduct levels during formation and the following removal over 15 weeks, using a modified Edman degradation and ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results demonstrated for the first time that the relatively high exposure during the intervention period was reflected in corresponding distinct increases of 2,3-diHOPr-Val levels in all participants, following the expected slope for hemoglobin adduct formation and removal over time. The mean adduct level increased from 4.0 to 12.2 pmol 2,3-diHOPr-Val/g hemoglobin. By using a nonlinear mixed model, values for the adduct level/dose ratio (k, mean 0.082 pmol 2,3-diHOPr-Val/g hemoglobin per µg glycidol/kg body weight) and the adduct lifetime (τ, mean 104 days, likely the lifetime of the erythrocytes) were determined. Interindividual variability was generally low. 2,3-DiHOPr-Val was therefore proven to be a biomarker of the external dietary exposure to fatty acid esters of glycidol. From the background adduct levels observed in our study, a mean external glycidol exposure of 0.94 µg/kg body weight was estimated. This value is considerably higher than current estimates for adults using occurrence and consumption data of food. Possible reasons for this discrepancy are discussed (other oral or inhalational glycidol sources, endogenous formation, exposure to other chemicals also forming the adduct 2,3-diHOPr-Val). Further research is necessary to clarify the issue.

Evaluation of the Edman degradation product of vancomycin bonded to core-shell particles as a new HPLC chiral stationary phase.

A modified macrocyclic glycopeptide-based chiral stationary phase (CSP), prepared via Edman degradation of vancomycin, was evaluated as a chiral selector for the first time. Its applicability was compared with other macrocyclic glycopeptide-based CSPs: TeicoShell and VancoShell. In addition, another modified macrocyclic glycopeptide-based CSP, NicoShell, was further examined. Initial evaluation was focused on the complementary behavior with these glycopeptides. A screening procedure was used based on previous work for the enantiomeric separation of 50 chiral compounds including amino acids, pesticides, stimulants, and a variety of pharmaceuticals. Fast and efficient chiral separations resulted by using superficially porous (core-shell) particle supports. Overall, the vancomycin Edman degradation product (EDP) resembled TeicoShell with high enantioselectivity for acidic compounds in the polar ionic mode. The simultaneous enantiomeric separation of 5 racemic profens using liquid chromatography-mass spectrometry with EDP was performed in approximately 3 minutes. Other highlights include simultaneous liquid chromatography separations of rac-amphetamine and rac-methamphetamine with VancoShell, rac-pseudoephedrine and rac-ephedrine with NicoShell, and rac-dichlorprop and rac-haloxyfop with TeicoShell.

Hemoglobin adducts of furfuryl alcohol in genetically modified mouse models: Role of endogenous sulfotransferases 1a1 and 1d1 and transgenic human sulfotransferases 1A1/1A2.

Furfuryl alcohol (FFA) is a heat-induced food contaminant. Conversion by sulfotransferases (SULT) yields 2-sulfoxymethylfuran, which is prone to react with DNA and proteins. In order to monitor the internal FFA exposure we developed a technique for the mass spectrometric quantification of the adduct N-((furan-2-yl)methyl)-valine (FFA-Val) after cleavage from the N-termini of hemoglobin. In the current study the method was applied to investigate the influence of different SULT forms on the adduct formation in wild-type mice and three genetically modified mouse models treated with FFA. Two lines were devoid of endogenous Sult1a1 or Sult1d1, while another mouse line carried a transgene of human SULT1A1/1A2 in the Sult1a1/1d1 double knockout background. The Sult1d1 knockout did not influence adduct formation, whereas the lack of Sult1a1 reduced mean FFA-Val levels by 80% and 58% in male and female mice, respectively, in comparison to FFA-treated wild-type mice. The levels of FFA-Val in the humanized mice were elevated by factors of 2.7 (males) and 2.2 (females) as compared to the wild-type, indicating that SULT1A1/1A2 play a central role for FFA bioactivation also in humans. The excellent correlation between adduct levels in hepatic DNA and hemoglobin (r2 = 0.97) indicated that 2-sulfoxymethylfuran of hepatic origin is sufficiently stable to enter circulation and pass the cellular membrane of erythrocytes. This is a prerequisite for the application of FFA-Val as a biomarker of internal FFA exposure.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening.

BACKGROUND: Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. METHODS: Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. RESULTS: Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4-C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. CONCLUSIONS: This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.(AU)

A hemoglobin adduct as a biomarker for the internal exposure to the rodent carcinogen furfuryl alcohol.

Furfuryl alcohol is a common food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. Its carcinogenic effect in rodents originates most likely from sulfotransferase (SULT)-catalyzed conversion into the mutagenic sulfate ester 2-sulfoxymethylfuran. In this study, a protein adduct biomarker was sought for the medium-term internal exposure to furfuryl alcohol. A UPLC-MS/MS screening showed that the adduct N-((furan-2-yl)methyl)-Val (FFA-Val) at the N-terminus of hemoglobin is a valid target analyte. The Val cleavage by fluorescein isothiocyanate-mediated Edman degradation yielded 3-fluorescein-1-(furan-2-ylmethyl)-5-(propan-2-yl)-2-thioxoimidazolidin-4-one (FFA-Val-FTH), which was characterized by 1H and 13C NMR spectroscopy. An isotope-dilution method for the quantification of FFA-Val-FTH by UPLC-MS/MS was developed. It was used to study the adduct formation in furfuryl alcohol-treated FVB/N mice and the influence of ethanol and the alcohol dehydrogenase (ADH) inhibitor 4-methylpyrazole on the adduct levels. The administration of 400 mg/kg body weight furfuryl alcohol alone led to 12.5 and 36.7 pmol FFA-Val/g Hb in blood samples of male and female animals, respectively. The co-administration of 1.6 g ethanol/kg body weight increased FFA-Val levels by 1.4-fold in males and by 1.5-fold in females. The co-administration of 100 mg 4-methylpyrazole/kg body weight had a similar effect on the adduct levels. A high correlation was observed between adduct levels in hemoglobin and in hepatic DNA samples determined in the same animal experiment. This indicated that FFA-Val is a valid biomarker for the internal exposure to 2-sulfoxymethylfuran, which may be suitable to monitor furfuryl alcohol exposure also in humans.

Photoaffinity Labeling of Pentameric Ligand-Gated Ion Channels: A Proteomic Approach to Identify Allosteric Modulator Binding Sites.

Photoaffinity labeling techniques have been used for decades to identify drug binding sites and to study the structural biology of allosteric transitions in transmembrane proteins including pentameric ligand-gated ion channels (pLGIC). In a typical photoaffinity labeling experiment, to identify drug binding sites, UV light is used to introduce a covalent bond between a photoreactive ligand (which upon irradiation at the appropriate wavelength converts to a reactive intermediate) and amino acid residues that lie within its binding site. Then protein chemistry and peptide microsequencing techniques are used to identify these amino acids within the protein primary sequence. These amino acid residues are located within homology models of the receptor to identify the binding site of the photoreactive probe. Molecular modeling techniques are then used to model the binding of the photoreactive probe within the binding site using docking protocols. Photoaffinity labeling directly identifies amino acids that contribute to drug binding sites regardless of their location within the protein structure and distinguishes them from amino acids that are only involved in the transduction of the conformational changes mediated by the drug, but may not be part of its binding site (such as those identified by mutational studies). Major limitations of photoaffinity labeling include the availability of photoreactive ligands that faithfully mimic the properties of the parent molecule and protein preparations that supply large enough quantities suitable for photoaffinity labeling experiments. When the ligand of interest is not intrinsically photoreactive, chemical modifications to add a photoreactive group to the parent drug, and pharmacological evaluation of these chemical modifications become necessary. With few exceptions, expression and affinity-purification of proteins are required prior to photolabeling. Methods to isolate milligram quantities of highly enriched pLGIC suitable for photoaffinity labeling experiments have been developed. In this chapter, we discuss practical aspects of experimental strategies to identify allosteric modulator binding sites in pLGIC using photoaffinity labeling.

An isotope-dilution UPLC-MS/MS technique for the human biomonitoring of the internal exposure to glycidol via a valine adduct at the N-terminus of hemoglobin.

Fatty acid esters of glycidol (glycidyl esters) are processing contaminants generated as a byproduct of the industrial deodorization of vegetable oils and fats. Oral intake of glycidyl esters leads to the release of glycidol in the gastrointestinal tract. Glycidol is carcinogenic, genotoxic and teratogenic in rodents. It is rated as probably carcinogenic to humans (IARC group 2A). The determination of internal exposure of glycidol may support the assessment of the possible human health risks related to glycidyl ester intake. For this purpose, hemoglobin adducts of glycidol may be suitable biomarkers reflecting the cumulative exposure of up to four months. We applied a modified Edman degradation to assess the glycidol adduct at the N-terminal valine, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), of hemoglobin. The modified valine was cleaved with fluorescein-5-isothiocyanate (FITC), resulting in the formation of N-(2,3-dihydroxypropyl)-valine fluorescein thiohydantoin (DHP-Val-FTH). An isotope-dilution technique was developed for the quantification of the thiohydantoin analyte by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and DHP-Val-d7-FTH as reference standard. The limit of detection was 4 fmol DHP-Val-FTH per injection corresponding to 0.7pmol 2,3-diHOPr-Val/g hemoglobin. The adduct levels in blood samples of 12 non-smoking participants were in the range of 2.2-4.9pmol 2,3-diHOPr-Val/g hemoglobin. The current work presents the first isotope-dilution technique using UPLC-MS/MS for the quantification of 2,3-diHOPr-Val at the N-terminus of hemoglobin as a sensitive and convenient alternative to earlier GC-MS methods.

On-resin N-terminal peptoid degradation: Toward mild sequencing conditions.

A novel approach to sequentially degrade peptoid N-terminal N-(substituted)glycine residues on the solid-phase using very mild conditions is reported. This method relies on the treatment of resin-bound, bromoacetylated peptoids with silver perchlorate in THF, leading to an intramolecular cyclization reaction to liberate the terminal residue as a N-substituted morpholine-2,5-dione, resulting in a truncated peptoid upon hydrolysis and a silver bromide byproduct. Side-chain functional group tolerance is explored and reaction kinetics are determined. In a series of pentapeptoids possessing variable, non-nucleophilic side-chains at the second position (R(2) ), we demonstrate that sequential N-terminal degradation of the first two residues proceeds in 87% and 74% conversions on average, respectively. We further demonstrate that the degradation reaction is selective for peptoids, and represents substantial progress toward a mild, iterative sequencing method for peptoid oligomers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 726-736, 2016.

Peptide purification using the chemoselective reaction between N-(methoxy)glycine and isothiocyanato-functionalized resin.

An efficient peptide purification strategy is established, comprising the selective reaction of an N-terminal N-(methoxy)glycine residue of the peptide and isothiocyanato-functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation-prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240-272). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

A novel N-terminal degradation reaction of peptides via N-amidination.

The cleavage of amide bonds requires considerable energy. It is difficult to cleave the amide bonds in peptides at room temperature, whereas ester bonds are cleaved easily. If peptide bonds can be selectively cleaved at room temperature, it will become a powerful tool for life science research, peptide prodrug, and tissue-targeting drug delivery systems. To cleave a specific amide bond at room temperature, the decomposition reaction of arginine methyl ester was investigated. Arginine methyl ester forms a dimer; the dimer releases a heterocyclic compound and ornithine methyl ester at room temperature. We designed and synthesized N-amidinopeptides based on the decomposition reaction of arginine methyl ester. Alanyl-alanine anilide was used as the model peptide and could be converted into N-degraded peptide, alanine anilide, via an N-amidination reaction at close to room temperature. Although the cleavage rate in pH 7.4 phosphate buffered saline (PBS) at 37°C was slow (t1/2=35.7h), a rapid cleavage rate was observed in 2% NaOH aq (t1/2=1.5min). To evaluate the versatility of this reaction, a series of peptides with Lys, Glu, Ser, Cys, Tyr, Val, and Pro residue at the N-terminal were synthesized; they showed rapid cleavage rates of t1/2 values from 1min to 10min.