Name: determination of the gas-phase acidities of oligopeptides
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<p class="jove_content"&gt; Le travail a été financé par la National Science Foundation (CHE-0749737). L&#39;utilisation de l&#39;instrument a été fourni par le Fonds pour la spectrométrie de masse à l&#39;Université du Pacifique.</p

1. Préparation des solutions d&#39;échantillon <ol><li> Préparer d&#39;abord les solutions mères du peptide et les six acides de référence en utilisant un solvant mixte de méthanol et d&#39;eau dans un rapport en volume de 1:1. Les solutions mères devraient avoir une concentration d&#39;environ 10 -3 M. </li><li> Peser 1 mg de l&#39;échantillon de peptide solide, A 3 CH, dans un tube Eppendorf de 1.5 ml et ajouter 1,0 ml de solvant mixte de méthanol et d&#39;eau, et mélanger à l&#39;a...

<ol>
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La mesure réussie de l&#39;acidité en phase gazeuse d&#39;un peptide repose en grande partie sur le choix des acides de référence appropriés. Les acides de référence idéales sont des composés organiques de structure similaire présentant des valeurs d&#39;acidité en phase gazeuse bien établies. Les acides de référence doivent avoir des structures semblables les uns aux autres. Cela permettra d&#39;assurer une entropie similaire de la déprotonation de chacun des acides de référence de l&#39;ensemble. Les ...

Les acidités de résidus d&#39;acides aminés sont parmi les plus importantes propriétés thermochimiques qui influent sur ​​les structures, la réactivité, et les processus de pliage-dépliage des protéines 9,19. Résidus d&#39;acides aminés individuels présentent souvent différentes acidités efficaces en fonction de leurs emplacements dans les protéines. En particulier, les résidus situés sur les sites actifs présentent souvent considérablement perturbé acidités. Un exemple est le résidu cystéine qui résident dans les sites actifs de la super-famille des enzymes 20,21 thiorédoxine. La cystéine du site actif est anormalement acide par rapport à celles de protéines dépliées 3-5. Il a été suggéré que la conformation hélicoïdale peut avoir une contribution significative à l&#39;acidité inhabituelle. Il ya de vastes études expérimentales sur les propriétés acide-base de peptides menées dans les solutions, en particulier dans des solutions aqueuses 2,6-8. Les résultats ont souvent été compliquées par les effets de solvant7. En fait, la plupart des sites actifs dans les protéines sont situés près de la zone intérieure où les effets de solvants sont réduites au minimum 9,10. Afin de comprendre les propriétés acido-basiques intrinsèques de peptides et de protéines, il est important de réaliser les études dans un environnement exempt de solvant. Nous présentons ici une méthode basée sur la spectrométrie de masse pour la détermination de l&#39;acidité en phase gazeuse. L&#39;approche est appelée la méthode cinétique Cooks prolongée. Cette méthode a été appliquée avec succès à un large éventail de systèmes chimiques pour la détermination des différentes propriétés thermodynamiques, telles que l&#39;acidité en phase gazeuse, l&#39;affinité protons, les ions de l&#39;affinité du métal, l&#39;affinité électronique et l&#39;énergie d&#39;ionisation 11-15, 22-26. Nous avons appliqué cette méthode pour déterminer les acidités en phase gazeuse d&#39;une série d&#39;oligo-cystéine polyalanine et la cystéine-polyglycine peptides 17,18,27. Ces études montrent que la cystéine N-terminal Peptides sont beaucoup plus acides que celles C-terminaux correspondants. Les acidités élevées de l&#39;ancien sont probablement dues à des effets de conformation hélicoïdale dont l&#39;anion thiolate est fortement stabilisée par l&#39;interaction avec l&#39;hélice macro-dipôle. En raison de la nature non-volatiles et thermolabiles de peptides, la méthode cinétique est l&#39;approche la plus pratique disponible à l&#39;heure actuelle pour produire de l&#39;acide-base des quantités thermochimiques raisonnablement précises de peptides 28. Le régime général et l&#39;équation associée à la méthode cinétique sont présentés dans la figure 1. La détermination de l&#39;acidité en phase gazeuse d&#39;un peptide (AH) commence par la formation d&#39;une série d&#39;anions de la grappe de protons liés, [A • h • A i] ¯ (ou [A ¯ • H + • A i ¯] ¯), dans la région de la source d&#39;ions du spectromètre de masse, où A et A i ¯ ¯ sont les formes déprotonés du peptide etles acides de référence, respectivement. Les acides de référence sont des composés organiques avec des acidités-en phase gazeuse connu. Les acides de référence doivent avoir des structures semblables à l&#39;autre (mais pas nécessairement similaire à celui du peptide). La similitude des structures entre les acides de référence assure la similitude des entropies de déprotonation parmi eux. Le cluster proton-bound anions sont choisis masse et par collision activés et ensuite dissocié en utilisant dissociation expériences induite par collision (CID) pour donner des anions monomères correspondants, A et A i ¯ ¯, avec des constantes de vitesse de k et k i, respectivement, à la figure 1a. Si fragmentations secondaires sont négligeables, le rapport d&#39;abondance des ions fragments CID, [A ¯] / [A i ¯], représente une mesure approximative du rapport des constantes de vitesse, k / k i. Dans l&#39;hypothèse où il n&#39;y a pas activÃ inverseles barrières d&#39;ions pour les deux canaux de dissociation, l&#39;ion produit CID rapports de branchement, ln [A ¯] / [A i ¯], sera linéairement corrélée à l&#39;acidité en phase gazeuse du peptide (Δ acide H) et ceux des acides de référence (acide Δ H i), comme le montre la figure 1b. Dans cette équation, l&#39;acide Δ H moyenne est de l&#39;acidité en phase gazeuse moyenne des acides de référence, Δ (Δ S) est le terme entropie (qui peut être supposée constante si les acides de référence sont structurellement similaires les unes aux autres), R est l&#39; constante de gaz universelle, et T eff est la température effective du système. La température effective est un paramètre empirique qui dépend de plusieurs variables expérimentales, dont l&#39;énergie de collision. La valeur de l&#39;acidité en phase gazeuse est déterminé par la construction de deux ensembles d&#39;emplacements thermo-cinétiques. La première série est obcontenues en traçant ln ([A ¯] / [A i ¯]) contre l&#39;acide Δ H i - acide Δ H moyenne, comme le montre la figure 4a. La régression linéaire donnera une série de lignes droites avec des pentes de X = 1 / RT eff et intercepte de Y = - [Δ acide H - acide Δ H avg] / RT eff - Δ (Δ S) / R. La deuxième série de parcelles est obtenue en traçant les intersections obtenues (Y) de la première série contre les pentes correspondantes (X), comme le montre la figure 4b. La régression linéaire produit une nouvelle ligne avec une pente de Δ acide H - acide Δ H moyenne et une interception de Δ (Δ S) / R. La valeur de Δ acide H est alors obtenue à partir de la pente et le terme entropie, Δ (Δ S), est obtenu à partirl&#39;interception. Les expériences sont réalisées à l&#39;aide d&#39;un spectromètre de masse quadripolaire triple relié à une ionisation par électrospray (ESI) de la source d&#39;ions. Un schéma de principe du spectromètre de masse est illustrée à la figure 2. Les expériences CID sont effectuées en masse sélectionner les anions de cluster protons liés avec la première unité de quadripolaire et en leur permettant subissent des collisions avec des atomes d&#39;argon fuites dans la chambre de collision qui est maintenue à une pression d&#39;environ 0,5 mTorr. Les ions de produit de dissociation de masse sont analysés avec la troisième unité de quadripôle. Les spectres CID sont enregistrés à plusieurs énergies de collision avec la gamme m / z suffisamment large pour couvrir tous les fragments secondaires possibles. Les intensités d&#39;ions sur les produits CID sont mesurées par l&#39;instrument de mise en surveillance de réaction choisie (SRM) du mode dans lequel le balayage est focalisé sur des ions de produit sélectionné. Les expériences CID sont effectués à quatre différentes énergies de collision, ce qui correspond àénergies centre de masse (E cm) de 1,0, 1,5, 2,0, et 2,5 eV, respectivement. L&#39;énergie dans le centre de masse est calculé en utilisant l&#39;équation: E cm = E laboratoire [m / (m + m)], où E laboratoire est l&#39;énergie de collision dans le référentiel du laboratoire, m est la masse de l&#39;argon, et M est la masse de l&#39;ion de grappe proton lié. Dans cet article, nous utilisons les oligopeptides Ala 3 CysNH 2 (A 3 CH) comme composé modèle. L&#39;extrémité C-terminale est amidée, et le groupe thiol (SH) du résidu de cystéine sera le site acide. La sélection des acides de référence appropriés est crucial pour le succès de mesure de l&#39;acidité en phase gazeuse. Les acides de référence idéales sont structurellement similaires (les unes aux autres) des composés organiques ayant des valeurs d&#39;acidité en phase gazeuse bien établies. Les acides de référence doivent avoir des valeurs d&#39;acidité à proximité de celle des peptides. Pour le peptide A 3 CH, six halogénés carboxyliqueacides C sont choisis comme les acides référence. Les six acides de référence sont l&#39;acide chloracétique (MCAH), l&#39;acide bromoacétique (MBAH), de l&#39;acide difluoroacétique (DFAH), l&#39;acide dichloroacétique (DCAH), de l&#39;acide dibromoacétique (DBAH), et trifluoroacétique (TFAH). Deux d&#39;entre eux, DFAH et MBAH, seront utilisés pour illustrer le protocole.

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		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>Mass Spectrometer</td>
			<td>Varian</td>
			<td>1200 L and 320 L</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>402923</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bromoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>B56307</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Difluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>142859</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>D54702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibromoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>242357</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of the gas-phase acidities of oligopeptides

Les résidus d&#39;acides aminés situés à des positions différentes dans les protéines pliées présentent souvent des degrés différents de acidités. Par exemple, un résidu cystéine situé dans ou près de l&#39;extrémité N-terminale d&#39;une hélice est souvent plus acide que celui au niveau ou près de l&#39;extrémité C-terminale de 1 à 6. Bien que de vastes études expérimentales sur les propriétés acide-base de peptides ont été réalisées dans la phase condensée, en particulier dans des solutions aqueuses 6-8, les résultats sont souvent compliquées par les effets de solvant 7. En fait, la plupart des sites actifs dans les protéines sont situés près de la zone intérieure où les effets de solvant ont été réduites au minimum 9,10. Afin de comprendre les propriétés acido-basiques intrinsèques de peptides et de protéines, il est important d&#39;effectuer des études dans un environnement exempt de solvant. Nous présentons une méthode pour mesurer les acidités des oligopeptides dans la phase gazeuse. Nous utilisons un contenant de la cystéine oligopeptides, Ala 3 CysNH <sUB&gt; 2 (CH 3 A), en tant que composé modèle. Les mesures sont basées sur la méthode Cooks prolongée bien établie cinétique (figure 1) 11-16. Les expériences sont réalisées à l&#39;aide d&#39;un spectromètre de masse quadripolaire triple interfacé avec une ionisation par électrospray (ESI) de la source d&#39;ions (figure 2). Pour chaque échantillon de peptide, de plusieurs acides de référence sont sélectionnées. Les acides de référence sont des composés organiques ayant une structure similaire acidités-en phase gazeuse connu. Une solution du mélange du peptide et un acide de référence est introduit dans le spectromètre de masse, et le groupe anionique en phase gazeuse proton lié d&#39;acide peptide-référence est formée. Le cluster proton-bound est masse isolée et ensuite fragmenté par dissociation expériences induite par collision (CID). Les abondances d&#39;ions fragments obtenus sont analysés en utilisant une relation entre les acidités et le cluster ion cinétique de dissociation. L&#39;acidité en phase gazeuse du peptide est ensuite obtenie par régression linéaire des parcelles thermo-cinétiques 17,18. La méthode peut être appliquée à une variété de systèmes moléculaires, y compris les composés organiques, les acides aminés et leurs dérivés, les oligonucléotides et oligopeptides. En comparant les acidités en phase gazeuse mesurées expérimentalement avec les valeurs calculées pour les différentes conformations, les effets conformationnels sur les acidités peuvent être évalués.

<ol><li> Les expériences de bracketing CID fournissent des informations sur les acidités relatives du peptide par rapport aux acides de référence sélectionnés. Deux représentant spectres CID du peptide (A 3 CH) avec deux acides de référence, DFAH et MBAH, sont présentés dans la figure 3. Dans la figure 3a l&#39;abondance d&#39;ions (hauteur de pic) de l&#39;ion peptidique est plus faible que celle de DFA ¯, et la figure 3b, l&#39;abondance ionique de l&#39;ion peptidique ...

Watch this Scientific Journal Video about Determination of the Gas-phase Acidities of Oligopeptides at JoVE.com

Determination of the Gas-phase Acidities of Oligopeptides

La détermination des acidités en phase gazeuse d&#39;oligopeptides contenant de la cystéine est décrite. Les expériences sont réalisées à l&#39;aide d&#39;un spectromètre de masse quadripolaire triple. Les acidités relatives des peptides sont mesurées à l&#39;aide d&#39;expériences de dissociation induite par collision, et les acidités quantitatives sont déterminées en utilisant les cuisiniers de longues méthode cinétique.

Détermination des acidités en phase gazeuse d&#39;oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

determination-of-the-gas-phase-acidities-of-oligopeptides

Research

JoVE Journal

Chemistry

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.&#160;

Rapides à haut débit amylose Détermination dans lyophilisé échantillons de pommes de terre de tubercules

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

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Chimie

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

From Constructs to Crystals &amp;#8211; Towards Structure Determination of &amp;#946;-barrel Outer Membrane Proteins

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

De Constructs à cristaux - Vers Structure Détermination de β-cylindre externe protéines membranaires

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

Détermination des groupes fonctionnels carbonylés dans Bio-huiles par titrage potentiométrique: La méthode Faix

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Determination of Thermodynamic Properties of Alkaline Earth-liquid Metal Alloys Using the Electromotive Force Technique

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline earth-liquid metal alloys as functions of both composition and temperature in order to acquire thermodynamic data. The cell consists of a chemically stable solid-state CaF2-AF2 electrolyte (where A is the alkaline-earth element such as Ca, Sr, or Ba), with binary A-B alloy (where B is the liquid metal such as Bi or Sb) working electrodes, and a pure A metal reference electrode. Emf data are collected over a temperature range of 723 K to 1,123 K in 25 K increments for multiple alloy compositions per experiment and the results are analyzed to yield activity values, phase transition temperatures, and partial molar entropies/enthalpies for each composition.

This protocol describes the measurement of the electromotive force of alkaline-earth elements in liquid metal alloys at high temperatures (723-1,123 K) to determine their thermodynamic properties, including activity, partial molar entropy, partial molar enthalpy, and phase transition temperatures, over a wide composition range.

Détermination des propriétés thermodynamiques des alliages de métaux alcalino-terreux-liquide en utilisant la Technique de Force électromotrice

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Démêler les fibres d’aramide de copolymère haute résistance afin de permettre la détermination de leurs propriétés mécaniques

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Synthesis and Structure Determination of &amp;#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Synthèse et détermination de Structure des isomères PIIIA µ-conotoxine avec disulfure différentes connectivités

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Techniques de spectrométrie de mobilité-masse d'ion pour déterminer la structure et les mécanismes de la reconnaissance d'ion de métal et de l'activité de Redox des Oligopeptides de liaison de métal

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they are utilized in a wide variety of applications. In most studies employing photoswitches, several important photophysical properties such as maximum wavelengths of absorption and emission, molar attenuation coefficient, fluorescence lifetime, and photoisomerization quantum yield are carefully determined to investigate their electronic states and transition processes. However, measurement of the photoisomerization quantum yield, the efficiency of photoisomerization with respect to the absorbed photons, in a typical laboratory setting is often complicated and prone to error because it requires the implementation of rigorous spectroscopic measurements and calculations based on an appropriate integration method. This article introduces a set of procedures to measure the photoisomerization quantum yield of a bistable photoswitch using a photochromic hydrazone. We anticipate that this article will be a useful guide for the investigation of bistable photoswitches that are being increasingly developed.

Photoisomerization quantum yield is a fundamental photophysical property that should be accurately determined in the investigation of newly developed photoswitches. Here, we describe a set of procedures to measure the photoisomerization quantum yield of a photochromic hydrazone as a model bistable photoswitch.

Détermination du rendement quantique de photoisomérisation d’un photocommutateur Hydrazone

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they ...

Analyse des pesticides dans l’avocat à l’aide de la méthode QuEChERS et de la CG-MS/MS

Determination of 45 Pesticides in Avocado Varieties by the QuEChERS Method and Gas Chromatography-Tandem Mass Spectrometry

Gas chromatography (GC) tandem mass spectrometry (MS/MS) stands as a preeminent analytical instrument extensively employed for the surveillance of pesticide residues in food. Nevertheless, these methods are vulnerable to matrix effects (MEs), which can potentially affect accurate quantification depending on the specific combination of analyte and matrix. Among the various strategies to mitigate MEs, matrix-matched calibration represents the prevailing approach in pesticide residue applications due to its cost-effectiveness and straightforward implementation. In this study, a total of 45 representative pesticides were analyzed in three different varieties of avocado (i.e., Criollo, Hass, and Lorena) using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate and GC-MS/MS. 
For this purpose, 5 g of the avocado sample was extracted with 10 mL of acetonitrile, and then 2.5 g of ammonium formate was added to induce phase separation. Subsequently, the supernatant underwent a cleanup process via dispersive solid-phase extraction employing 150 mg of anhydrous MgSO4, 50 mg of primary-secondary amine, 50 mg of octadecylsilane, 10 mg of graphitized carbon black, and 60 mg of a zirconium oxide-based sorbent (Z-Sep+). The GC-MS/MS analysis was successfully performed in less than 25 min. Rigorous validation experiments were carried out to assess the performance of the method. The examination of a matrix-matched calibration curve for each variety of avocado revealed that the ME remained relatively consistent and less than 20% (considered as a soft ME) for most pesticide/variety combinations. Furthermore, the method&#180;s limits of quantification were lower than 5 &#181;g/kg for all three varieties. Finally, the recovery values for most pesticides fell within the acceptable range of 70-120%, with relative standard deviation values below 20%.

Préparation d’échantillons d’avocat à l’aide de la méthode QuEChERS avec du formiate d’ammonium pour l’analyse de pesticides

The present protocol describes the analysis of multiclass pesticide residues in avocado varieties using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate, followed by gas chromatography-tandem mass spectrometry.