Name: determination of the gas-phase acidities of oligopeptides
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<p class="jove_content"&gt; Arbejdet blev støttet af National Science Foundation (CHE-0.749.737). Instrumentet forbrug blev leveret af massespektrometri Facility på University of the Pacific.</p

1.. Klargøring af prøven Solutions <ol><li> Først forberede stamopløsninger af peptidet og seks referencepunkter syrer ved hjælp af en blandet opløsningsmiddel af methanol og vand i et 1:1 volumenforhold. Stamopløsningerne bør have en koncentration på ca 10 -3 M. </li><li> Afvej 1 mg af den faste peptid prøve, A 3 CH, i et 1,5 ml Eppendorf-rør, og der tilsættes 1,0 ml blandet opløsningsmiddel af methanol og vand, og blandes med en hvirvel. </li><li> Afvej 1 mg difluoreddikesyre (DFAH...

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Den vellykkede måling af gas-fase surhedsgrad af et peptid i høj grad baseret på udvælgelsen af ​​egnede referencepunkter syrer. Den ideelle reference-syrer er strukturelt lignende organiske forbindelser med veletablerede gasfaseeksperimenter surhedsgrad værdier. Referencetallene syrer bør have lignende strukturer til hinanden. Dette vil sikre en tilsvarende entropi deprotonering for hver referenceopløsning syrer i sættet. Referenceværdierne syrer skal have surhedsgrad værdier tæt på de af peptider. For ...

De syrer af aminosyrerester er blandt de vigtigste termokemiske egenskaber, der påvirker strukturer, reaktivitet, og den folde-udfoldning processer af proteiner 9,19. Individuelle aminosyrerester ofte viser forskellige effektive syrer afhængigt af deres placeringer i proteiner. Ofte især resterne placeret på de aktive steder udviser signifikant forstyrret syrer. Et sådant eksempel er cysteinresten bosiddende i de aktive steder af thioredoxin super-familie af enzymer 20,21. Den aktive sted cystein er usædvanligt sure sammenlignet med dem i udfoldede proteiner 3-5. Det er blevet foreslået, at den spiralformede konformation kan have et væsentligt bidrag til den usædvanlige syreindhold. Der er omfattende eksperimentelle undersøgelser af syre-base egenskaber af peptider udføres i opløsninger, især i vandige opløsninger 2,6-8. Resultaterne blev ofte kompliceret af solventeffekter7.. Faktisk er de fleste af de aktive steder i proteiner placeret nær den indre område hvor solventeffekter minimeres 9,10. For at forstå iboende syre-base egenskaber af peptider og proteiner, er det vigtigt at udføre undersøgelserne i et opløsningsmiddel-frit miljø. Her introducerer vi et massespektrometri-baseret metode til bestemmelse af den gas-fasen surhedsgrad. Den fremgangsmåde er benævnt udvidede Cooks kinetiske metode. Denne metode er blevet anvendt med succes til en bred vifte af kemiske systemer til bestemmelse af forskellige termokemiske egenskaber, såsom gas-fase syreindhold, proton affinitet, metalionen affinitet, elektron affinitet, og ioniseringsenergi 11-15, 22-26. Vi har anvendt denne metode til at bestemme gas-fase syrer af en serie af oligo cysteinholdige polyalanin og cystein-polyglycin peptider 17,18,27. Disse undersøgelser viser, at den N-terminale cystein peptides er betydeligt mere sure end de tilsvarende C-terminale dem. De høje syrer i det tidligere skyldes sandsynligvis de spiralformede konformationelle virkninger hvor thiolat anion kraftigt stabiliseret ved interaktion med helix makro-dipol. På grund af den ikke-flygtige og termisk labile karakter af peptider, er den kinetiske metode den mest praktiske fremgangsmåde til rådighed på nuværende tidspunkt at fremstille rimeligt nøjagtige syre-base termokemiske mængder af peptider 28. Den generelle opbygning og ligningen forbundet med den kinetiske metode er vist i figur 1.. Bestemmelsen af gas-fasen surhedsgrad et peptid (AH) starter med dannelsen af en række proton-bundne klynge anioner, [A • H • A i] ¯ (eller [A ¯ • H + • Ai ¯] ¯), i ionkilden region i massespektrometret, hvor A ¯ og A i ¯ er deprotonerede former af peptidet ogreference syrer, hhv. Referenceværdierne syrer er organiske forbindelser med kendte gas-fase syrer. Referencetallene syrer skal have strukturer, der svarer til hinanden (men ikke nødvendigvis svarer til peptidet). Ligheden mellem strukturer mellem reference-syrer sikrer ligheden mellem de entropier af deprotonation blandt dem. Proton-bundne klynge anioner masse udvalgt og collisionally aktiveret og derefter dissocieret ved hjælp kollision-induceret dissociation (CID) eksperimenter til opnåelse af de tilsvarende monomere anioner, A ¯ og A i ¯, med hastighedskonstanterne for k og k I, henholdsvis, er vist i figur 1a. Hvis sekundære fragmenteringer er ubetydelige, den overflod forholdet mellem CID fragmentioner, [A ¯] / [A i ¯], udgør et omtrentligt mål for forholdet mellem ratekonstanter, k / k I. Under den antagelse, at der ikke er omvendt aktiveretion barrierer for både dissociation kanaler, CID produkt ion forgreningsforhold, ln [A ¯] / [A i. ¯], vil blive lineært korreleret til gasfase-surhed af peptid (Δ syre H) og de ​​af reference-syrer (Δ syre H i), som vist i figur 1b. I denne ligning er Δ acid H avg den gennemsnitlige gas-fase surhedsgrad reference syrer, Δ (Δ S) er den entropi sigt (hvilket kan antages konstant, hvis reference syrer er strukturelt ligner hinanden), R er universel gas konstant, og T eff er den effektive temperatur af systemet. Den effektive temperatur er en empirisk parameter, der afhænger af flere eksperimentelle variabler, herunder kollisionen energi. Værdien af ​​gasfase-syreindhold bestemmes ved at konstruere to sæt termo-kinetiske plots. Det første sæt er obopretholdt ved at plotte ln ([A ¯] / [A i ¯]) mod Δ acid H i - Δ syre H avg, som vist i figur 4a. Lineær regression vil give et sæt af lige linjer med skråningerne af X = 1 / RT EFF og aflytninger af Y = - [Δ acid H - Δ acid H avg] / RT EFF - Δ (Δ S) / R. Det andet sæt af grunde opnås ved at plotte de resulterende opfanger (Y) fra det første sæt mod de tilsvarende skråninger (X), som vist i figur 4b. Lineær regression producerer en ny linje med en hældning på Δ acid H - Δ acid H avg og en skæring med Δ (Δ S) / R. Værdien af Δ acid H opnås derefter fra hældningen og entropi sigt Δ (Δ S), fås fraskæringspunktet. Eksperimenterne udføres ved hjælp af en tripel kvadrupol massespektrometer grænseflader til en elektrospray (ESI) ionkilde. En skematisk diagram af massespektrometer er vist i figur 2.. De CID forsøg udføres efter masse at vælge proton-bundne klynge anioner med den første kvadrupol enheden og tillade dem at gennemgå kollisioner med argonatomer lækket ind kollisionen kammer, som holdes ved et tryk på omkring 0,5 mTorr. Dissociationen produkt ioner masse analyseres med den tredje quadrupol enheden. CID spektre optages på flere kollision energier med m / z interval bred nok til at dække alle mulige sekundære fragmenter. CID produkt ionintensiteter måles ved at sætte instrumentet i den valgte reaktion overvågning (SRM) tilstand, hvor scanningen er fokuseret på udvalgte produktområder ioner. CID eksperimenter udføres på fire forskellige kollision energier, svarende tilcenter-of-masse energi (E cm) på 1,0, 1,5, 2,0, og 2,5 eV, hhv. Centrum-of-masse energi beregnes ved ligning: E cm = E lab [m / (M + m)], hvor E lab er kollision energi i laboratoriet rammen, m er massen af argon og M er massen af ​​proton-bundne klynge ion. I denne artikel bruger vi oligopeptidet Ala 3 CysNH 2 (A 3 CH) som model compound. C-terminalen er amideret, og thiolgruppe (SH) i cysteinresten vil være den sure side. Udvælgelsen af ​​egnede referenceprodukter syrer er afgørende for en vellykket måling af gas-fasen surhedsgrad. Den ideelle reference-syrer er strukturelt ligner (på hinanden) organiske forbindelser med veletablerede gasfaseeksperimenter surhedsgrad værdier. Referenceværdierne syrer skal have surhedsgrad værdier tæt på at af peptider. For peptid A 3 CH, halogenerede seks carboxylic syrer er valgt som reference syrer. De seks referencepunkter syrer er chloreddikesyre (MCAH), bromeddikesyre (MBAH), difluoreddikesyre (DFAH), dichloreddikesyre (DCAH), dibromoacetic syre (DBAH), og trifluoreddikesyre (TFAH). To af dem, vil DFAH og MBAH, anvendes til at illustrere protokollen.

<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

Aminosyrerester placeret på forskellige positioner i foldede proteiner ofte udviser forskellige grader af syrer. For eksempel er en cysteinrest placeret ved eller nær N-terminalen af en helix ofte mere sure end ved eller nær C-terminalen 1-6. Selv omfattende eksperimentelle undersøgelser af syre-base egenskaber af peptider er blevet foretaget i den kondenserede fase, navnlig i vandige opløsninger 6-8, er resultaterne ofte kompliceret af solventeffekter 7. Faktisk er de fleste af de aktive steder i proteiner placeret nær den indre region, hvor opløsningsmiddel virkninger er blevet minimeret 9,10. For at forstå iboende syre-base egenskaber af peptider og proteiner, er det vigtigt at udføre undersøgelserne i et opløsningsmiddel-frit miljø. Vi præsenterer en metode til at måle syrer af oligopeptider i gasfase. Vi bruger en cystein-holdige oligopeptid, Ala 3 CysNH <sub&gt; 2 (A 3 CH), som modelforbindelse. Målingerne er baseret på veletablerede udvidet Cooks kinetisk metode (Figur 1) 11-16. Forsøgene udføres under anvendelse af en triple-kvadrupol massespektrometer interface med en elektrospray (ESI) ionkilde (figur 2). For hvert peptid stikprøve flere referencepunkter syrer valgt. Referenceværdierne syrer er strukturelt lignende organiske forbindelser med kendte gas-fase syrer. En opløsning af blandingen af ​​peptidet og en reference syre indføres i massespektrometret, og en gas-fase proton-bundne anioniske klynge af peptid-henvisning Syren dannes. Proton-bundet cluster masse isoleres og efterfølgende fragmenteret via kollision-induceret dissociation (CID) eksperimenter. Det resulterende fragment ion mængderne er analyseret ved en relation mellem syrer og klyngen ion dissociation kinetik. Gasfase-surhedsgrad peptidet er derefter obtaiNed ved lineær regression af termo-kinetiske plots 17,18. Fremgangsmåden kan anvendes til en bred vifte af molekylære systemer, herunder organiske forbindelser, aminosyrer og deres derivater, oligonukleotider og oligopeptider. Ved at sammenligne gas-fase syrer målt eksperimentelt med disse beregnede værdier for forskellige afarter kan konformationelle virkninger på syrer evalueres.

<ol><li> CID bracketing eksperimenter give oplysninger om de relative syrer af peptidet i forhold til de valgte referencestoffer syrer. To repræsentative CID spektre af peptidet (A 3 CH) med to reference syrer, DFAH og MBAH, er vist i figur 3.. I figur 3a ion overflod (tophøjde) af peptidet ion er svagere end DFA ¯, og i figur 3b, ion overflod af peptidet ion er stærkere end MBA ¯. De to spektre tyder på, at gasfase-surhedsgrad peptidet er i intervallet mellem syrer af disse to...

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Determination of the Gas-phase Acidities of Oligopeptides

Fastsættelsen af ​​gas-fase syrer af cystein-holdige oligopeptider beskrives. Eksperimenterne udføres ved hjælp af en tripel kvadrupol massespektrometer. De relative syrer af peptiderne måles ved hjælp kollision-induceret dissociation eksperimenter, og de kvantitative syrer bestemmes ved hjælp af de udvidede Cooks kinetisk metode.

Bestemmelse af gas-fase syrer af Oligopeptider

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

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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 Amylose Bestemmelse i frysetørret kartoffelknolden Prøver

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 ...

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.

Fra konstruktioner på Crystals - Mod Structure Bestemmelse af β-tønde ydermembranproteiner

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.

Bestemmelse af carbonylfunktionelle grupper i Bio-olier ved potentiometrisk titrering: 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 ...

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.

Bestemmelse af termodynamiske egenskaber af Alkaline jord-væske metallegeringer ved hjælp af elektromotoriske kraft teknik

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.

Udrede højstyrke Copolymer Aramid fibre til bestemmelse af mekaniske egenskaber

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.

Syntese og struktur bestemmelse af µ-Conotoxin PIIIA isomerer med forskellige disulfid Connectivities

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.

Ion Mobility-massespektrometri teknikker til bestemmelse af strukturen og mekanismerne for metal Iongenkendelse og Redoxaktivitet af metal bindende oligopeptider

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.

Bestemmelse af fotoisomeriseringskvanteudbyttet af en hydrazone-fotoswitch

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

Analyse af pesticider i avocado ved hjælp af QuEChERS-metoden og 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%.

Forberedelse af avocadoprøve ved hjælp af QuEChERS-metoden med ammoniumformiat til pesticidanalyse

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.