Name: determination of the gas-phase acidities of oligopeptides
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<p class="jove_content"&gt; Il lavoro è stato sostenuto dal National Science Foundation (CHE-0.749.737). L&#39;utilizzo dello strumento è stata fornita dalla spettrometria di massa Struttura presso la University of the Pacific.</p

1. Preparazione delle soluzioni campione <ol><li> Prima preparare le soluzioni madre del peptide e gli acidi riferimento sei usando un solvente misto di metanolo e acqua in un rapporto in volume di 1:1. Le soluzioni madre dovrebbero avere una concentrazione di circa 10 -3 M. </li><li> Pesare 1 mg del campione peptide solido, A 3 CH, in una provetta Eppendorf 1,5 ml e aggiungere 1,0 ml di solvente misto di metanolo e acqua, e miscelare con un vortice. </li><li> Pesare 1 mg di acido difluoroacetic ...

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La misurazione di successo della acidità in fase gassosa di un peptide basa in gran parte sulla selezione di acidi riferimento adeguati. Gli acidi riferimento ideali sono composti organici strutturalmente simili con valori di acidità in fase gas ben consolidati. Gli acidi riferimento dovrebbero avere strutture simili tra loro. Ciò garantirà un simile entropia di deprotonazione per ciascuno degli acidi riferimento del set. Gli acidi di riferimento devono avere valori di acidità prossimi a quelli dei peptidi. Per bre...

Le acidità di residui di aminoacidi sono tra i più importanti proprietà termochimici che influenzano le strutture, la reattività, e dei processi di piegatura-unfolding di proteine ​​9,19. Residui di singoli aminoacidi mostrano spesso diverse acidità efficaci a seconda delle loro posizioni in proteine. In particolare, i residui presenti in altri siti attivi presentano spesso perturbato significativamente acidità. Un esempio è il residuo di cisteina che risiedono nei siti attivi della tioredossina super-famiglia di enzimi 20,21. Il sito attivo è insolitamente cisteina acido rispetto a quelle in dispiegato proteine ​​3-5. È stato suggerito che la conformazione elicoidale può avere un contributo significativo alla acidità insolito. Ci sono ampi studi sperimentali sulle proprietà acido-base di peptidi effettuati in soluzioni, specialmente in soluzioni acquose 2,6-8. I risultati sono stati spesso complicati da effetti solvente7. Infatti, la maggior parte dei siti attivi di proteine ​​si trovano vicino alla regione interna dove effetti del solvente sono minimizzati 9,10. Per comprendere intrinseche proprietà acido-base di peptidi e proteine, è importante realizzare gli studi in un ambiente privo di solventi. Qui si introduce un metodo di spettrometria di massa a base per la determinazione dell&#39;acidità in fase gas. L&#39;approccio è indicato come il metodo cinetico Cuochi esteso. Questo metodo è stato applicato con successo per una vasta gamma di sistemi chimici per la determinazione delle varie proprietà termochimici, come l&#39;acidità in fase gassosa, l&#39;affinità protonica, lo ione metallico affinità, l&#39;affinità elettronica, e l&#39;energia di ionizzazione 11-15, 22-26. Abbiamo applicato questo metodo per determinare le acidità in fase gas di una serie di oligo-cisteina-cisteina e polialanine polyglycine peptidi 17,18,27. Questi studi dimostrano che la cisteina N-terminale peptidees sono significativamente più acido di quello dei corrispondenti quelli C-terminale. Le alte acidità delle prime sono probabilmente dovuti agli effetti conformazionali elicoidali in cui l&#39;anione tiolato è fortemente stabilizzato dalla interazione con l&#39;elica macro-dipolo. A causa della natura non volatile e termicamente labile di peptidi, il metodo cinetico è l&#39;approccio più pratico attualmente disponibili per produrre ragionevolmente accurate acido-base termochimici quantità di peptidi 28. Nel regime generale e l&#39;equazione associato al metodo cinetico sono mostrati in Figura 1. La determinazione dell&#39;acidità in fase gassosa di un peptide (AH) inizia con la formazione di una serie di cluster di anioni protone-bound, [A • H • A i] ¯ (o [A + H ¯ • • A i ¯] ¯), nella regione di sorgente ionica dello spettrometro di massa, dove A e A i ¯ ¯ sono le forme deprotonate del peptide egli acidi di riferimento, rispettivamente. Gli acidi di riferimento sono composti organici con noti acidità in fase gas. Gli acidi riferimento dovrebbero avere strutture analoghe tra loro (ma non necessariamente simile a quella del peptide). La somiglianza delle strutture tra acidi riferimento assicura la somiglianza dei entropie di deprotonazione tra loro. Il cluster protone-bound anioni vengono selezionati e massa collisionally attivati ​​e successivamente dissociato usando dissociazione (CID) esperimenti di collisione indotti a cedere gli anioni corrispondenti monomeriche, A e A i ¯ ¯, con rate costanti di K e K i, rispettivamente, mostrata in Figura 1a. Se frammentazioni secondarie sono trascurabili, il rapporto di abbondanza dei frammenti ionici CID, [A ¯] / [A i ¯], rappresenta una misura approssimativa del rapporto tra le costanti di velocità, k / k i. Partendo dal presupposto che non ci sono attivata inverso, [A °] / [A I °], verrà linearmente correlati all&#39;acidità fase gas del peptide (Δ acido H) e quelli degli acidi riferimento ln barriere per entrambi i canali ionici dissociazione, il CID ione prodotto branching ratio (acido Δ H i), come mostrato in Figura 1b. In questa equazione, acido Δ H avg è la media acidità in fase gassosa degli acidi riferimento, Δ (Δ S) è il termine entropia (che può essere assunta costante se gli acidi riferimento sono strutturalmente simili tra loro), R è la costante universale dei gas, e T eff è la temperatura effettiva del sistema. La temperatura efficace è un parametro empirico che dipende da diverse variabili sperimentali, inclusi l&#39;energia di collisione. Il valore di acidità in fase gassosa è determinato disegnando due serie di trame termo-cinetici. La prima serie è obcontenute tracciando ln ([A ¯] / [A i ¯]) contro l&#39;acido Δ H i - acido Δ H avg, come mostrato nella figura 4a. La regressione lineare produrrà una serie di linee rette con le piste di X = 1 / RT eff e intercetta di Y = - [Δ H acido - acido Δ H avg] / RT eff - Δ (Δ S) / R. La seconda serie di trame viene ottenuta riportando le intercettazioni risultanti (Y) con la prima serie contro le corrispondenti piste (X), come mostrato in Figura 4b. La regressione lineare produce una nuova linea con una pendenza di Δ H acido - acido Δ H avg e un&#39;intercettazione di Δ (Δ S) / R. Il valore di Δ acido H si ottiene poi dal pendio e il termine entropia, Δ (Δ S), è ottenuto dal&#39;intercetta. Gli esperimenti vengono eseguiti utilizzando uno spettrometro di massa triplo quadrupolo interfacciato ad un ionizzazione elettrospray (ESI) sorgente di ioni. Un diagramma schematico dello spettrometro di massa è mostrato in Figura 2. Gli esperimenti vengono eseguiti CID in massa selezionando gli anioni del cluster protone-bound con la prima unità quadrupolo e permettendo loro di sottoporsi collisioni con atomi di argon fuoriusciti nella camera di collisione mantenuto ad una pressione di circa 0,5 mTorr. Gli ioni prodotti di dissociazione di massa sono analizzati con la terza unità quadrupolo. Gli spettri CID sono registrati in diverse energie di collisione con il range m / z sufficientemente ampia da coprire tutte le possibili frammenti secondari. I CID intensità di ioni prodotti vengono misurate impostando lo strumento nel monitoraggio reazione selezionato (SRM) modalità in cui la scansione è focalizzata sulla ioni prodotti selezionati. Gli esperimenti CID vengono eseguite in quattro diverse energie di collisione, che corrispondono aenergie centro-di-massa (E cm) di 1.0, 1.5, 2.0, e 2.5 eV, rispettivamente. L&#39;energia centro-di-massa è calcolata usando l&#39;equazione: E = E cm lab [m / (M + m)], dove E è l&#39;energia laboratorio collisione nel sistema del laboratorio, m è la massa di argon, e M è la massa dello ione cluster di protone-bound. In questo articolo, si usa il oligopeptide Ala 3 CysNH 2 (A 3 CH) come composto modello. Il C-terminale è amidate e il gruppo tiolo (SH) del residuo cisteina sarà il sito acida. La selezione degli acidi riferimento adeguati è fondamentale per la misurazione riuscita dell&#39;acidità fase gas. Gli acidi riferimento ideali sono strutturalmente simili (tra loro) composti organici con valori di acidità in fase gas ben consolidati. Gli acidi di riferimento devono avere valori di acidità prossime a quella dei peptidi. Per il peptide A 3 CH, sei alogenati carboxyliacidi c sono scelti come gli acidi di riferimento. Gli acidi di riferimento sei sono cloroacetico (MCAH), acido bromoacetico (Mbah), acido difluoroacetic (DFAH), acido dicloroacetico (DCAH), acido dibromoacetic (dban), e trifluoroacetico (TFAH). Due di loro, DFAH e Mbah, saranno utilizzati per illustrare il protocollo.

<table border="1">
		<tbody>
		 <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

Residui amminoacidici situati in posizioni differenti in proteine ​​ripiegate spesso mostrano diversi gradi di acidità. Per esempio, un residuo di cisteina situato in prossimità o al N-terminale di un elica è spesso più acido che in prossimità o al C-terminale 1-6. Sebbene ampi studi sperimentali sulle proprietà acido-base di peptidi sono stati effettuati nella fase condensata, in particolare in soluzioni acquose 6-8, i risultati sono spesso complicati da effetti solvente 7. Infatti, la maggior parte dei siti attivi di proteine ​​si trovano vicino alla regione interna in cui sono stati minimizzati effetti del solvente 9,10. Per comprendere intrinseche proprietà acido-base di peptidi e proteine, è importante effettuare gli studi in un ambiente privo di solventi. Presentiamo un metodo per misurare le acidità degli oligopeptidi in fase gas. Usiamo una cisteina contenente oligopeptide, Ala 3 CysNH <sub&gt; 2 (A 3 CH), come composto modello. Le misurazioni sono basate sul metodo cinetico esteso Cuochi consolidata (Figura 1) 11-16. Gli esperimenti vengono eseguiti usando uno spettrometro di massa triplo quadrupolo interfacciato con ionizzazione elettrospray (ESI) sorgente di ioni (Figura 2). Per ogni campione peptide, sono selezionati diversi acidi riferimento. Gli acidi di riferimento sono composti organici strutturalmente simili con noti acidità in fase gas. Una soluzione della miscela del peptide e un acido riferimento viene introdotta nello spettrometro di massa, ed una fase gassosa protone-bound gruppo anionico di acido-peptide di riferimento è formato. Il cluster protone-bound è massa isolata e successivamente frammentata tramite dissociazione (CID) esperimenti di collisione-indotte. I risultanti abbondanze di ioni frammento vengono analizzati utilizzando un rapporto tra le acidità e il cluster di ioni dissociazione cinetica. L&#39;acidità fase gas del peptide è poi ottenito da regressione lineare delle trame termo-cinetica 17,18. Il metodo può essere applicato ad una varietà di sistemi molecolari, compresi i composti organici, amminoacidi e loro derivati, oligonucleotidi e degli oligopeptidi. Confrontando le acidità fase gas misurati sperimentalmente con quei valori calcolati per i diversi conformeri, effetti conformazionali sui acidità possono essere valutati.

<ol><li> Gli esperimenti bracketing CID forniscono informazioni sui relativi acidità del peptide rispetto agli acidi riferimento selezionati. Due rappresentativo spettri CID del peptide (A 3 CH) con due acidi riferimento, DFAH e Mbah, sono mostrati in Figura 3. In Figura 3a all&#39;abbondanza ione (altezza picco) del peptide ione è più debole di quella del DFA ¯, e in Figura 3b, l&#39;abbondanza ione dello ione peptide è più forte di quello del MBA ¯. I due spettri suggeriscon...

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Determination of the Gas-phase Acidities of Oligopeptides

La determinazione delle acidità fase gas di oligopeptidi contenenti cisteina è descritto. Gli esperimenti vengono eseguiti utilizzando uno spettrometro di massa a triplo quadrupolo. Le acidità relative dei peptidi sono valutate con esperimenti di dissociazione indotta da collisione, e le acidità quantitativi sono determinati utilizzando i Cuochi estesi metodo cinetico.

Determinazione delle acidità in fase gassosa di oligopeptidi

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

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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;

Rapid High Throughput amilosio Determinazione in liofilizzati campioni dei tuberi

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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Chimica

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.

Da costrutti a cristalli - Verso Struttura Determinazione della β-canna esterna proteine ​​di membrana

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.

Determinazione del carbonile gruppi funzionali in Bio-oli da potenziometrica Titolazione: Il Metodo 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.

Determinazione delle proprietà termodinamiche di leghe di metalli alcalino-terrosi-liquido usando la tecnica di forza elettromotrice

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.

Districante fibre di aramide copolimero ad alta resistenza per consentire la determinazione delle loro proprietà meccaniche

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.

Sintesi e determinazione della struttura di µ-conotossina PIIIA isomeri con bisolfuro diverse connettività

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.

Tecniche di spettrometria ione Mobilità-Massa per determinare la struttura e i meccanismi del riconoscimento degli ioni metallici e dell'attività di redox degli oligopeptidi di metal linciazione

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.

Determinazione della resa quantistica di fotoisomerazione di un interruttore foto Hydrazone

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

Analisi dei pesticidi nell'avocado utilizzando il metodo QuEChERS e 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%.

Preparazione del campione di avocado utilizzando il metodo QuEChERS con formiato di ammonio per l'analisi dei pesticidi

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.