Name: determination of the gas-phase acidities of oligopeptides
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<p class="jove_content"&gt; Die Arbeit wurde von der National Science Foundation (CHE-0749737) unterstützt. Das Instrument wurde von der Nutzung Massenspektrometrie Einrichtung an der University of the Pacific vorgesehen.</p

1. Vorbereitung der Musterlösungen <ol><li> Bereiten Sie zuerst die Stammlösungen des Peptids und die sechs Referenz Säuren Verwendung eines gemischten Lösungsmittels aus Methanol und Wasser in einem Volumenverhältnis 1:1. Die Stammlösungen sollte eine Konzentration von etwa 10 -3 M. </li><li> Man wiegt 1 mg des festen peptid-Probe, A 3 CH, in ein 1,5 ml Eppendorf-Röhrchen und 1,0 ml Lösungsmittelgemisch aus Methanol und Wasser, und Mischen mit einem Vortex. </li><li> Abwiegen 1 mg Difluo...

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Die erfolgreiche Messung der Gasphase Säuregehalt eines Peptids weitgehend beruht auf der Auswahl geeigneter Referenz Säuren. Die ideale Nachschlagewerk Säuren sind strukturell ähnliche organische Verbindungen mit etablierten Gasphase Säurewerte. Die Referenz-Säuren sollten ähnliche Strukturen zueinander haben. Dadurch wird eine ähnliche Entropie Deprotonierung für jeden der Referenz-Säuren in dem Satz. Die Referenz-Säuren sollten Säurewerte nahe an denen der Peptide. Für kürzere Cystein enthaltenden Oligo...

Die Aciditäten Aminosäurereste gehören zu den wichtigsten thermochemischen Eigenschaften, die die Strukturen zu beeinflussen, die Reaktivität und die Faltungs-Entfaltungs-Prozesse von Proteinen 9,19. Einzelne Aminosäurereste zeigen oft unterschiedliche effektive Aciditäten je nach ihren Standorten in Proteinen. Insbesondere die Reste an den aktiven Zentren befinden zeigen oft deutlich gestört Aciditäten. Ein solches Beispiel ist der Cysteinrest mit Wohnsitz in den aktiven Zentren des Thioredoxin super-Familie von Enzymen 20,21. Das aktive Zentrum Cystein ist ungewöhnlich sauren im Vergleich zu denen in entfaltete Proteine ​​3-5. Es wurde vorgeschlagen, dass die Helix-Konformation kann einen wesentlichen Beitrag zur ungewöhnlichen Säure haben. Es gibt umfangreiche experimentelle Untersuchungen über die Säure-Base-Eigenschaften von Peptiden in Lösungen durchgeführt, insbesondere in wässrigen Lösungen 2,6-8. Die Ergebnisse wurden oft von Lösungsmitteleffekte kompliziert7. In der Tat sind die meisten der aktiven Stellen in Proteinen in der Nähe des inneren Bereichs in dem Lösungsmittel 9,10 Wirkungen minimiert befinden. Um intrinsische Säure-Base-Eigenschaften von Peptiden und Proteinen zu verstehen, ist es wichtig für die Durchführung der Untersuchungen in einer lösungsmittelfreien Umgebung. Hier stellen wir eine Massenspektrometrie-basierte Verfahren zur Bestimmung der Gasphase Säure. Der Ansatz wird als erweiterte Cooks kinetische Methode bezeichnet. Diese Methode wurde erfolgreich in einer Vielzahl von chemischen Systemen zur Bestimmung von verschiedenen thermochemischen Eigenschaften, wie z. B. der Gasphase Säure, die Protonen-Affinität, die Affinität für Metallionen, die Elektronenaffinität und der Ionisierungsenergie 11-15 angelegt, 22-26. Wir haben diese Methode, um die Gas-Phase Aciditäten einer Reihe von Oligo-Cystein und Cystein-Polyalanin Polyglycin Peptide 17,18,27 bestimmen angewendet. Diese Studien zeigen, dass die N-terminalen Cystein Peptides sind deutlich saurer als die entsprechenden C-terminalen diejenigen. Die hohe Acidität der ersteren sind wahrscheinlich auf die schraubenförmigen Konformationseffekte in dem das Thiolat-Anion stark durch die Wechselwirkung mit der Wendel Makro-Dipol stabilisiert wird. Aufgrund der nicht-flüchtige und thermisch instabil sind Peptide, die kinetische Methode ist es am praktischsten derzeit vorliegenden einigermaßen genaue Säure-Base-thermochemischen Mengen von Peptiden 28 zu erzeugen. Das allgemeine Schema und die Gleichung mit der kinetischen Verfahren zusammenhängen, sind in 1 gezeigt. Die Bestimmung der Gasphase Säure eines Peptids (AH) mit der Bildung einer Reihe von Protonen-gebundenen Clusteranionen [A • h • A i] ¯ (oder [A ¯ • H + • A ¯ i] ¯), in der Ionenquelle Region des Massenspektrometers, wobei A ¯ A ¯ i sind die deprotonierte Form des Peptids unddie Referenz-Säuren sind. Die Referenz-Säuren sind organische Verbindungen mit bekannten Gasphase Aciditäten. Die Säuren dienen sollten Strukturen einander ähnlich (jedoch nicht notwendigerweise ähnlich der des Peptids). Die Ähnlichkeit der Strukturen zwischen Referenz-Säuren stellt die Ähnlichkeit der Entropien Deprotonierung unter ihnen. Das Proton-gebundene Cluster Anionen Masse ausgewählt und collisionally aktiviert und anschließend mit dissoziierten Stoßfragmentierung (CID) Experimente, um die entsprechenden monomeren Anionen ergeben, A und A ¯ ¯ i, mit Geschwindigkeitskonstanten k und k i, jeweils in Abbildung 1a. Wenn sekundäre Fragmentierungen vernachlässigbar sind, die Fülle Verhältnis der CID-Fragment-Ionen, [A ¯] / [A i ¯], stellt ein ungefähres Maß für das Verhältnis der Geschwindigkeitskonstanten k / k i. Unter der Annahme, dass es keine umgekehrte aktivierendenIonensperren sowohl Dissoziationskanäle die CID Produkt Ion Verzweigungsverhältnisse, ln [A ¯] / [A i ¯], linear auf die Gasphasen-Acidität des Peptids (Δ Säure H) und diejenigen werden korreliert werden die Referenz-Säuren (Δ Säure H i), wie in 1b gezeigt. In dieser Gleichung Δ Säure H avg die durchschnittliche Gasphase Säuregehalt der Referenz-Säuren ist, Δ (Δ S) die Entropie Begriff (das kann als konstant angenommen werden, wenn die Referenz-Säuren sind strukturell einander ähnlich) ist, R die universelle Gaskonstante und T eff die effektive Temperatur des Systems. Die effektive Temperatur ist eine empirische Parameter ist, der mehrere experimentelle Variablen, einschließlich der Kollisionsenergie abhängt. Der Wert der Gasphase Säure wird erreicht, indem zwei Sätze von thermo-kinetische Diagramme bestimmt. Der erste Satz der OBenthaltenen durch Auftragung ln ([A ¯] / [A i ¯]) gegen Δ Säure H i - Δ Säure H avg, wie in Abbildung 4a gezeigt. Lineare Regression wird eine Reihe von geraden Linien mit den Pisten von X = 1 / RT eff und fängt von Y Ausbeute = - [Δ Säure H - Δ Säure H avg] / RT eff - Δ (Δ S) / R. Der zweite Satz von Flächen durch Auftragen der resultierenden fängt (Y) aus dem ersten Satz mit den entsprechenden geneigten Flächen (X), wie in 4b gezeigt ist. Lineare Regression erzeugt eine neue Linie mit einer Steigung von Δ Säure H - Δ Säure H avg und ein Abschnitt von Δ (Δ S) / R. Der Wert Δ H Säure wird dann aus der Steigung erhalten, und die Entropieterm, Δ (Δ S), erhält man ausder Schnittpunkt. Die Experimente werden unter Verwendung eines Dreifach-Quadrupol-Massenspektrometer eine Schnittstelle mit einer Elektrospray-Ionisations (ESI)-Ionenquelle. Ein schematisches Diagramm des Massenspektrometers ist in Abbildung 2 dargestellt. Die CID Experimente Masse Auswahl der Protonen-bound-Anionen mit dem ersten Quadrupol-Einheit und es ihnen ermöglicht, um Kollisionen mit Argon-Atomen in der Kollision Kammer, die bei einem Druck von etwa 0,5 mTorr gehalten wird zugespielt Eingriff durchgeführt. Die Spaltprodukt Ionen Masse mit dem dritten Quadrupol-Gerät analysiert. Die CID-Spektren werden bei mehreren Kollisionsenergien mit dem m / z-Bereich breit genug, um alle möglichen sekundären Fragmente decken aufgezeichnet. Die CID Produkt Ionenintensitäten indem das Gerät in der gewählten Reaktionstemperatur Monitoring (SRM), in dem die Scan ausgewählten Produkt-Ionen fokussiert wird gemessen. Die CID Experimente werden an vier verschiedenen Kollisionsenergien durchgeführt, entsprechendCenter-of-mass Energien (E cm) von 1,0, 1,5, 2,0 und 2,5 eV. Die Mitte-der-Masse-Energie wird unter Verwendung der Gleichung: E = E Labor cm [m / (M + m)], wobei E Labor die Aufprallenergie im Laborsystem ist, m die Masse der Argon und M die Masse des Proton-gebundenen Cluster-Ionen. In diesem Artikel verwenden wir die Oligo-Ala 3 CysNH 2 (A 3 CH) als Vorbild Verbindung. Der C-Terminus amidiert ist und die Thiolgruppe (SH) des Cysteins wird die saure Website. Die Auswahl eines geeigneten Referenz Säuren ist von entscheidender Bedeutung für die erfolgreiche Messung der Gasphase Säure. Die ideale Nachschlagewerk Säuren sind strukturell ähnlich (miteinander) organische Verbindungen mit etablierten Gasphase Säurewerte. Die Säuren dienen sollte Säure Werte nahe, dass der Peptide. Für das Peptid A 3 CH, halogenierte sechs carbonsäurec Säuren werden als Referenz-Säuren gewählt. Die sechs Referenz Säuren sind Chloressigsäure (MCAH) Bromessigsäure (MBAH), Difluoressigsäure (DFAH), Dichloressigsäure (DCAH), dibromoacetic Säure (DBAH) und Trifluoressigsäure (TFAH). Zwei von ihnen, wird DFAH und MBAH, verwendet werden, um das Protokoll zu veranschaulichen.

<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

Aminosäurereste an verschiedenen Positionen in gefalteter Proteine ​​liegen häufig eine unterschiedliche Grade der Acidität. Zum Beispiel ist ein Cystein-Rest am oder nahe dem N-Terminus einer Wendel liegt oft saurer als das bei oder in der Nähe des C-Terminus 1-6. Obwohl umfangreiche experimentelle Untersuchungen über die Säure-Base-Eigenschaften von Peptiden wurden in der kondensierten Phase, insbesondere in wässrigen Lösungen 6-8 durchgeführt, werden die Ergebnisse oft durch Lösungsmitteleffekte 7 kompliziert. In der Tat sind die meisten der aktiven Stellen in Proteinen in der Nähe des inneren Bereichs, wo Lösungsmittel-Effekte wurden 9,10 haben minimiert wird. Um intrinsische Säure-Base-Eigenschaften von Peptiden und Proteinen zu verstehen, ist es wichtig, die Studien in einer lösungsmittelfreien Umgebung durchzuführen. Wir präsentieren eine Methode, um die Aciditäten Oligopeptide in der Gasphase zu messen. Wir verwenden eine Cystein enthaltenden Oligo-, Ala 3 CysNH <sub&gt; 2 (A 3 CH), als Modell Verbindung. Die Messungen werden auf dem gut etablierten erweiterten Cooks kinetische Methode (Abbildung 1) 11-16 bezogen. Die Experimente werden unter Verwendung einer Dreifach-Quadrupol-Massenspektrometer eine Schnittstelle mit einer Elektrospray-Ionisations (ESI)-Ionenquelle (Abbildung 2). Für jedes Peptid Beispiel werden mehrere Referenz Säuren. Die Referenz-Säuren sind strukturell ähnliche organische Verbindungen mit bekannten Gasphase Aciditäten. Eine Lösung der Mischung aus dem Peptid und einem Referenz Säure in das Massenspektrometer eingeführt wird, und ein Gas-Phasen Proton-gebundene anionische Gruppe von Peptid-Referenz Säure gebildet wird. Das Proton-gebundene Cluster Masse isoliert und anschließend über Stoßfragmentierung (CID) Experimente fragmentiert. Die entstehenden Fragmentionen Häufigkeiten werden analysiert unter Verwendung einer Beziehung zwischen der Acidität und Clusterion Dissoziationskinetik. Das Gasphasen-Acidität des Peptids wird dann obtainiert durch lineare Regression der thermo-kinetischen Grundstücke 17,18. Das Verfahren kann auf eine Vielzahl von molekularen Systemen, einschließlich organischer Verbindungen, Aminosäuren und deren Derivate, Oligonukleotide und Oligopeptide eingesetzt werden. Durch den Vergleich der Gasphase Acidität experimentell mit den Werten für verschiedene Konformere berechnet gemessen wird, kann Auswirkungen auf die Konformation Acidität ausgewertet werden.

<ol><li> Die CID Bracketing Experimente liefern Informationen über die relativen Acidität des Peptids im Vergleich zu den ausgewählten Referenz-Säuren. Zwei repräsentative CID-Spektren des Peptids (A 3 CH) mit zwei Referenz-Säuren, DFAH und MBAH, sind in Abbildung 3 dargestellt. In Fig. 3a ist der Ionenhäufigkeit (Peakhöhe) des Peptid-Ion ist schwächer als die des DFA ¯ und in 3b ist die Ionenhäufigkeit des Peptids Ionen stärker als die des MBA ¯. Die beiden Spektren zei...

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

Die Bestimmung der Gasphase Acidität von Cystein enthaltenden Oligopeptide beschrieben. Die Experimente werden unter Verwendung eines Dreifach-Quadrupol-Massenspektrometer. Die relativen Aciditäten der Peptide werden mit Stoßfragmentierung Experimente, und die quantitative Aciditäten werden unter Verwendung der erweiterten Cooks kinetische Methode.

Bestimmung der Gas-Phase Aciditäten von 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;

Schnelle High Throughput Amylose Bestimmung in Gefriergetrocknete Potato Tuber Proben

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 ...

Forschung

Chemie

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.

Von Konstrukten Crystals - Auf dem Weg zu Strukturbestimmung von β-Fass-Außenmembranproteine

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.

Bestimmung der Carbonylfunktionen in Bio-Öle durch potentiometrische Titration: Die Faix Methode

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.

Bestimmung der thermodynamischen Eigenschaften von Alkaline Erde-Liquid Metall-Legierungen mit der elektromotorischen Kraft-Technik

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.

Entwirren hohe Festigkeit-Copolymer-Aramid-Fasern um die Bestimmung der mechanischen Eigenschaften ermöglichen

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.

Synthese und Strukturaufklärung von µ-vor PIIIA Isomere mit verschiedenen Disulfid Verschaltungen

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.

Ionen-Mobilitäts-Massenspektrometrie-Techniken zur Bestimmung der Struktur und Mechanismen der Metallionenerkennung und Redox-Aktivität von Metallbindungsoligopeptiden

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.

Bestimmung der Photoisomerisierungsquantenausbeute eines Hydrazon-Photoschalters

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

Analyse von Pestiziden in Avocados mit der QuEChERS-Methode und 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%.

Probenvorbereitung von Avocados nach der QuEChERS-Methode mit Ammoniumformiat für die Pestizidanalytik

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.