Name: determination of the gas-phase acidities of oligopeptides
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<p class="jove_content"&gt; Arbeidet ble støttet av National Science Foundation (CHE-0749737). Instrumentet bruk ble gitt av massespektrometri Facility ved University of the Pacific.</p

En. Tilberedning av prøven Solutions <ol><li> Tilberedes først stamløsninger av peptid og de seks referanse RNA ved bruk av en blandet oppløsningsmiddel av metanol og vann i et volumforhold 01:01. De stamløsninger bør ha en konsentrasjon på omtrent 10 -3 M. </li><li> Vei opp 1 mg av den faste prøven peptid, A 3 CH, i et 1,5 ml Eppendorf-rør og tilsett 1,0 ml av blandet oppløsningsmiddel av metanol og vann, og blandes ved hjelp av en vortex. </li><li> Vei opp 1 mg Difluoreddiksyre (DFAH)...

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Den vellykkede måling av gassfase-surhet av et peptid avhengig i stor grad av valget av egnede syrer referanse. Den ideelle referanse syrer er strukturelt ligner organiske forbindelser med veletablerte gass-fase surhet verdier. Referansepunktene syrer bør ha lignende strukturer til hverandre. Dette vil sikre en lignende entropi av deprotonering for hvert av referanse syrer i settet. Referansepunktene syrer bør ha surhet verdier nær dem av peptidene. For kortere cystein-inneholdende oligopeptider med amidert C-termin...

De acidities av aminosyrer er blant de viktigste termokjemiske egenskaper som påvirker strukturene, den reaktivitet, og folding-utfolder prosesser av proteiner 9,19. Individuelle aminosyrer ofte viser ulike effektive acidities avhengig av deres steder i proteiner. Spesielt rester ligger i de aktive setene viser ofte betydelig perturberte acidities. Ett slikt eksempel er cysteinrest bosatt i de aktive områder av thioredoxin super-familien av enzymer 20,21. Den aktive sete cystein er unormalt surt forhold til de i utfoldet proteiner 3-5. Det har vært antydet at det skruelinjeformede konformasjon kan ha et betydelig bidrag til den uvanlige surhet. Det er omfattende eksperimentelle studier på syre-base-egenskaper av peptider gjennomføres i oppløsninger, spesielt i vandige oppløsninger 2,6-8. Resultatene ble ofte komplisert av løsemiddel effekter7. Faktisk er mesteparten av de aktive seter i proteiner lokalisert nær den indre område hvor løsemiddel effekter minimaliseres 9,10. For å forstå iboende syre-base-egenskaper av peptider og proteiner, er det viktig å utføre studiene i et oppløsningsmiddel-fritt miljø. Her introduserer vi en massespektrometri-basert metode for bestemmelse av gass-fase surhet. Denne tilnærmingen er omtalt som den utvidede Cooks kinetisk metode. Denne metoden har blitt vellykket anvendt for et bredt utvalg av kjemiske systemer for bestemmelse av forskjellige termokjemiske egenskaper, så som gassfase-surhet, proton affinitet, metallionet affinitet, elektronet affinitet, og ioniseringsenergien 11-15, 22-26. Vi har brukt denne metoden for å bestemme gass-fase acidities av en serie med oligo cystein-polyalanine og cystein-polyglycine peptider 17,18,27. Disse studiene viser at N-terminal cystein peptides er betydelig surere enn de tilsvarende C-terminale seg. De høye acidities av det tidligere er trolig på grunn av de spiralformede konformasjonsforandringer effekter der tiolatanion er sterkt stabilisert av samspillet med helix makro-dipol. På grunn av den ikke-flyktige og termisk labile naturen av peptider, er den kinetiske metode den mest praktiske tilnærmingen finnes idag for å produsere rimelig nøyaktig syre-base termokjemiske mengder av peptidene 28. Den generelle ordningen og ligningen forbundet med den kinetiske metode er vist på figur 1. Fastsettelsen av gass-fase surhet av et peptid (AH) starter med dannelsen av en serie av proton-bundet cluster anioner, [A • H • En i] ¯ (eller [A ¯ • H + • Å i ¯] ¯), i ionekilden region av massespektrometer, hvor A og A i ¯ ¯ er deprotonert former av peptidet ogreferanse syrer, henholdsvis. De referanse syrer er organiske forbindelser med kjente gass-fase acidities. Referansepunktene syrer bør ha strukturer som ligner hverandre (men ikke nødvendigvis lik den for peptid). Likheten av strukturene mellom referanse syrer sikrer likheten av entropies av deprotonering blant dem. Proton-bundet klynge anioner er valgt masse og collisionally aktivert og deretter dissosiert med sammenstøt-indusert dissosiasjon (CID) eksperimenter for å gi det tilsvarende monomere anioner, A og A i ¯ ¯, med hastighetskonstanter av K og k i, henholdsvis, er vist i figur 1a. Dersom sekundære fragmentations er ubetydelige, overflod forholdet av CID-fragment-ioner, [A ¯] / [Å i ¯], representerer et tilnærmet mål for forholdet mellom hastighetskonstantene, k / k i. Under forutsetning av at det ikke er omvendt aktiion barrierer for både dissosiasjon kanaler, CID produkt ion forgreningsforhold, Ln [A •] / [Å i ¯], vil bli lineært korrelert til gassfase-surhet av peptidet (Δ syre H) og de ​​av referanse-syrer (Δ syre H-i), som vist i figur 1b. I denne ligningen er Δ syre H avg gjennomsnittlig gass-fase surhet av referanse syrer, er Δ (Δ S) entropi sikt (som kan antas konstant dersom referanse syrer er strukturelt lik hverandre), er R universelle gasskonstanten og T eff er den effektive temperaturen i systemet. Den effektive temperatur er en empirisk parameter som avhenger av flere eksperimentelle variabler, inkludert kollisjonsenergien. Verdien av gassfaseprosessen surhet bestemmes ved å konstruere to sett av termo-kinetiske plott. Det første settet er obskje ved å plotte ln ([A ¯] / [A i ¯]) mot Δ syre H i - Δ syre H gj.snitt, som vist i figur 4a. Lineær regresjon vil gi et sett med rette linjer med bakkene i X = 1 / RT eff og fanger av Y = - [Δ syre H - Δ syre H avg] / RT eff - Δ (Δ S) / R. Det andre settet av tomter blir oppnådd ved å plotte de resulterende avskjærer (y) fra det første sett mot de tilsvarende bakker (X), som vist i figur 4b. Lineær regresjon produserer en ny linje med en helling på Δ syre H - Δ syre H avg og en fange av Δ (Δ S) / R. Verdien av Δ syre H blir deretter oppnådd fra helling og entropi sikt, Δ (Δ S), innhentesskjæringspunktet. Forsøkene er utført ved hjelp av en trippel kvadrupol massespektrometer sammenknyttet med en elektrospray ionisering (ESI) ionekilde. Et skjematisk diagram av massespektrometer er vist i figur 2.. De CID eksperimenter utføres etter masse å velge de proton-bundet cluster anioner med den første kvadrupol enhet og gi dem muligheten til å gjennomgå kollisjoner med argon atomer lekket inn i kollisjonen kammer som er holdt ved et trykk på rundt 0,5 mTorr. De dissosiasjon produktet ioner er masse analysert med den tredje kvadrupol enhet. CID spektra er registrert på flere kollisjon energier med m / z serien bred nok til å dekke alle mulige sekundære fragmenter. De CID produkt ion intensiteter måles ved å sette instrumentet i den valgte reaksjon overvåking (SRM) modus der skanningen er fokusert på utvalgte produktområdene ioner. De CID eksperimenter utføres på fire forskjellige kollisjon energier, tilsvarendecenter-of-masse energi (E cm) på 1,0, 1,5, 2,0, og 2,5 eV, henholdsvis. Senter-til-masse energien beregnes ved hjelp av ligningen: E = E cm lab [M / (M + m)], hvor E er lab kollisjonsenergien i laboratoriet ramme, m er massen av argon, og M er massen av den proton-bundet klynge ion. I denne artikkelen bruker vi oligopeptide Ala 3 CysNH 2 (A 3 CH) som modell sammensatte. C-terminalen er amidert, og tiolgruppe (SH) av cystein-rester vil være det sure område. Valget av egnet referanse syrer er avgjørende for en vellykket måling av gassfase-surhet. Den ideelle referanse syrer er strukturelt lik (til hverandre) organiske forbindelser med veletablerte gass-fase surhet verdier. Referansepunktene syrer bør ha surhet verdier nær at av peptider. For peptid A 3 CH, halogenerte seks carboxylic syrer er valgt som referanse-syrer. De seks referanse syrer er kloreddiksyre (MCAH), bromeddiksyre (mbah), Difluoreddiksyre (DFAH), dikloreddiksyreanhydrid syre (DCAH), dibromoacetic syre (DBAH), og trifluoreddiksyre (TFAH). To av dem, vil DFAH og mbah, benyttes til å illustrere den protokoll.

<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

Aminosyre rester ligger på forskjellige posisjoner i foldede proteiner ofte viser ulike grader av acidities. For eksempel er en cysteinrest lokalisert ved eller nær N-enden av en heliks ofte mer surt enn det på eller nær C-terminus 1-6. Selv om omfattende eksperimentelle studier på syre-base-egenskaper av peptidene er blitt gjennomført i den kondenserte fase, spesielt i vandige oppløsninger 6-8 er resultatene ofte komplisert av løsemiddel effekter 7. Faktisk er mesteparten av de aktive seter i proteiner lokalisert nær den indre område hvor løsemiddel effekter har blitt minimalisert 9,10. For å forstå iboende syre-base-egenskaper av peptider og proteiner, er det viktig å utføre studiene i et oppløsningsmiddel-fritt miljø. Vi presenterer en metode for å måle acidities av oligopeptider i gass-fase. Vi bruker en cystein-holdig oligopeptide, Ala 3 CysNH <sub&gt; 2 (A 3 CH), slik modellen forbindelsen. Målingene er basert på de veletablerte utvidet Cooks kinetisk metode (figur 1) 11-16. Forsøkene er utført ved hjelp av en trippel-kvadrupol massespektrometer tilkobles med en electrospray ionisering (ESI) ion kilde (figur 2). For hvert peptid prøve, blir flere referansepunkter syrer valgt. De referanse syrer er strukturelt ligner organiske forbindelser med kjente gass-fase acidities. En oppløsning av blandingen av peptidet og et referanse-syre innføres i massespektrometeret, og en gass-fase proton-bundet anionisk klynge av peptid-referanse syre dannes. Proton-bundet klyngen er masse isolert og deretter fragmentert via kollisjon-indusert dissosiasjon (CID) eksperimenter. De resulterende fragment ion abundances er analysert ved hjelp av et forhold mellom acidities og klyngen ion dissosiasjon kinetikk. Den gass-fase surhet av peptidet er da obtainert ved lineær regresjon av termo-kinetiske tomter 17,18. Fremgangsmåten kan anvendes på en rekke ulike molekylære systemer, inklusive organiske forbindelser, aminosyrer og deres derivater, oligonukleotider og oligopeptider. Ved å sammenligne de gass-fase acidities målt eksperimentelt med de verdiene som er beregnet for forskjellige conformers kan konformasjonsendringer virkninger på acidities evalueres.

<ol><li> De CID bracketing eksperimenter gi informasjon om de relative acidities av peptid i forhold til de valgte referanse syrer. To representative CID-spektra av peptidet (A CH 3) med to referanse-syrer, DFAH og mbah, er vist i figur 3.. I figur 3a ione overflod (topphøyde) av peptidet ion er svakere enn den av DFA ¯, og i figur 3b er det ion overflod av peptidet ion sterkere enn MBA ¯. De to spektra antyder at gassfase-surheten av peptid er i størrelsesorden mellom acidities ...

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

Bestemmelsen av gassfase-acidities av cystein-inneholdende oligopeptider er beskrevet. Forsøkene er utført ved hjelp av en trippel kvadrupol massespektrometer. De relative acidities av peptidene blir målt ved hjelp av kollisjon-indusert dissosiasjon eksperimenter, og de kvantitative acidities fastlegges ved hjelp av utvidede Cooks kinetisk metode.

Bestemmelse av gass-fase Acidities av 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

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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 høy gjennomstrømning Amylose Bestemmelse i Freeze Tørket potet 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 ...

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 konstruerer Crystals - Mot Struktur Bestemmelse av β-fat ytre membranproteiner

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.

Fastsettelse av Karbonyl funksjonelle grupper i bio-oljer av Potensiometrisk 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.

Fastsettelse av termodynamisk egenskaper av alkalisk jord-flytende metall legeringer med Elektromotorisk kraft-teknikk

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.

Disentangling høy styrke Copolymer Aramid fibre å aktivere fastsettelse av sine mekaniske egenskaper

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 proteinstrukturer av μ-Conotoxin PIIIA isomerene med forskjellige Disulfide 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-Mass massespektrometri teknikker for fastsettelse av struktur og mekanismer for metal ion Recognition og Redox aktivitet av metall bindende Oligopeptides

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 av Photoisomerization Quantum Yield av en Hydrazone Photoswitch

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

Analysere plantevernmidler i avokado ved hjelp av 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%.

Avokadoprøveforberedelse ved bruk av QuEChERS-metoden med ammoniumformiat for plantevernmiddelanalyse

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.