Name: determination of the gas-phase acidities of oligopeptides
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<p class="jove_content"&gt; Arbetet stöddes av National Science Foundation (CHE-0.749.737). Instrumentet användningen lämnades av masspektrometri Facility vid University of the Pacific.</p

Ett. Beredning av provet Solutions <ol><li> Först förbereder stamlösningarna av peptiden och de sex syror Referens Använd ett blandat lösningsmedel av metanol och vatten i ett 01:01 volymförhållande. De stamlösningar bör ha en koncentration av ca 10 -3 M. </li><li> Väg upp ett mg av den fasta peptiden prov, A 3 CH, i ett 1,5 ml Eppendorf-rör och tillsätt 1,0 ml av blandat lösningsmedel av metanol och vatten, och blanda med hjälp av en virvel. </li><li> Väg upp ett mg av difluorätt...

<ol>
	<li>Forsyth, W. R., Antosiewicz, J. M., Robertson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empirical+relationships+between+protein+structure+and+carboxyl+pKa+values+in+proteins.">Empirical relationships between protein structure and carboxyl pKa values in proteins.</a> Proteins: Struct. Funct. Genet. 48 (2), 388-403 (2002).</li><li>Huyghues-Despointes, B. M. P., Scholtz, J. M., Baldwin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+single+aspartate+on+helix+stability+at+different+positions+in+a+neutral+alanine-based+peptide.">Effect of a single aspartate on helix stability at different positions in a neutral alanine-based peptide.</a> Protein Sci. 2 (10), 1604-1611 (1993).</li><li>Takahashi, N., Creighton, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Reactivity+and+Ionization+of+the+Active+Site+Cysteine+Residues+of+Escherichia+coli+Thioredoxin.">On the Reactivity and Ionization of the Active Site Cysteine Residues of Escherichia coli Thioredoxin.</a> Biochemistry. 35 (25), 8342-8353 (1996).</li><li>Gan, Z. R., Sardana, M. K., Jacobs, J. W., Polokoff, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+thioltransferase+-+the+active+site+cysteines+display+differential+reactivity.">Yeast thioltransferase - the active site cysteines display differential reactivity.</a> Archives of Biochemistry and Biophysics. 282 (1), 110-115 (1990).</li><li>Philipps, B., Glockshuber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomization+of+the+Entire+Active-site+Helix+alpha+1+of+the+Thiol-disulfide+Oxidoreductase+DsbA+from+Escherichia+coli.">Randomization of the Entire Active-site Helix alpha 1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli.</a> J. Biol. Chem. 277 (45), 43050-43057 (2002).</li><li>Joshi, H. V., Meier, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+a+peptide+helix+macrodipole+on+the+pKa+of+an+Asp+side+chain+carboxylate.">The effect of a peptide helix macrodipole on the pKa of an Asp side chain carboxylate.</a> J. Am. Chem. Soc. 118, 12038-12044 (1996).</li><li>Kortemme, T., Creighton, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionization+of+cysteine+residues+at+the+termini+of+model+&#945;-helical+peptides.+Relevance+to+unusual+thiol+pKa+values+in+proteins+of+the+thioredoxin+family.">Ionization of cysteine residues at the termini of model &#945;-helical peptides. Relevance to unusual thiol pKa values in proteins of the thioredoxin family.</a> J. Mol. Biol. 253 (5), 799-812 (1995).</li><li>Gallo, E. A., Gellman, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+C-Terminal+Cationic+Group+on+the+Competition+between+&#945;-Helical+Turn+and+&#946;-Turn+in+a+Model+Depsipeptide.">Effect of a C-Terminal Cationic Group on the Competition between &#945;-Helical Turn and &#946;-Turn in a Model Depsipeptide.</a> J. Am. Chem. Soc. 116 (25), 11560-11561 (1994).</li><li>Honig, B., Nicholls, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+electrostatics+in+biology+and+chemistry.">Classical electrostatics in biology and chemistry.</a> Science. 268 (5214), 1144-1149 (1995).</li><li>Warshel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrostatic+basis+of+structure-function+correlation+in+proteins.">Electrostatic basis of structure-function correlation in proteins.</a> Acc. Chem. Res. 14 (9), 284-290 (1981).</li><li>Cooks, R. G., Patrick, J. S., Kotiaho, T., McLuckey, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermochemical+determinations+by+the+kinetic+method.">Thermochemical determinations by the kinetic method.</a> Mass Spectrom. Rev. 13 (4), 287-339 (1994).</li><li>Cooks, R. G., Koskinen, J. T., Thomas, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinetic+method+of+making+thermochemical+determinations.">The kinetic method of making thermochemical determinations.</a> J. Mass Spectrom. 34 (2), 85-92 (1999).</li><li>Cheng, X., Wu, Z., Fenselau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collision+Energy+Dependence+of+Proton-Bound+Dimer+Dissociation:+Entropy+Effects,+Proton+Affinities,+and+Intramolecular+Hydrogen-Bonding+in+Protonated+Peptides.">Collision Energy Dependence of Proton-Bound Dimer Dissociation: Entropy Effects, Proton Affinities, and Intramolecular Hydrogen-Bonding in Protonated Peptides.</a> J. Am. Chem. Soc. 115 (11), 4844-4848 (1993).</li><li>Cerda, B. A., Wesdemiotis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Li+,+Na+,+and+K++Binding+to+the+DNA+and+RNA+Nucleobases.+Bond+Energies+and+Attachment+Sites+from+the+Dissociation+of+Metal+Ion-Bound+Heterodimers.">Li+, Na+, and K+ Binding to the DNA and RNA Nucleobases. Bond Energies and Attachment Sites from the Dissociation of Metal Ion-Bound Heterodimers.</a> J. Am. Chem. Soc. 118 (47), 11884-11892 (1996).</li><li>Cooks, R. G., Wong, P. S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Method+of+Making+Thermochemical+Determinations:+Advances+and+Applications.">Kinetic Method of Making Thermochemical Determinations: Advances and Applications.</a> Acc. Chem. Res. 31 (7), 379-386 (1998).</li><li>Armentrout, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entropy+Measurements+and+the+Kinetic+Method:+a+Statistically+Meaningful+Approach.">Entropy Measurements and the Kinetic Method: a Statistically Meaningful Approach.</a> J. Am. Soc. Mass Spectrom. 11 (5), 371-379 (2000).</li><li>Ren, J., Tan, J. P., Harper, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-Phase+Acidities+of+Cysteine-Polyalanine+Peptides+I:+A3,4CSH+and+HSCA3,4.">Gas-Phase Acidities of Cysteine-Polyalanine Peptides I: A3,4CSH and HSCA3,4.</a> J. Phys. Chem. A. 113 (41), 10903-10912 (2009).</li><li>Morishetti, K. K., Huang, B. D. S., Yates, J. M., Ren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-Phase+Acidities+of+Cysteine-Polyglycine+Peptides:+The+Effect+of+the+Cysteine+Position.">Gas-Phase Acidities of Cysteine-Polyglycine Peptides: The Effect of the Cysteine Position.</a> Journal of the American Society for Mass Spectrometry. 21 (4), 603-614 (2010).</li><li>Fersht, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structure and mechanism in protein science. , (1999).</li><li>Martin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioredoxin-a+fold+for+all+reasons.">Thioredoxin-a fold for all reasons.</a> Structure. 3 (3), 245-250 (1995).</li><li>Carvalho, A. P., Fernandes, P. A., Ramos, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+Differences+in+the+Thioredoxin+Superfamily.">Similarities and Differences in the Thioredoxin Superfamily.</a> Progress in Biophysics &amp; Molecular Biology. 91 (3), 229-248 (2006).</li><li>Bouchoux, G., Sablier, M., Berruyer-Penaud, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+Thermochemical+Data+by+the+Extended+Kinetic+Method.">Obtaining Thermochemical Data by the Extended Kinetic Method.</a> J. Mass Spectrom. 39 (9), 986-997 (2004).</li><li>Bouchoux, G., Desaphy, S., Bourcier, S., Malosse, C., Bimbong, R. N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-Phase+Protonation+Thermochemistry+of+Arginine.">Gas-Phase Protonation Thermochemistry of Arginine.</a> J. Phys. Chem. B. 112 (11), 3410-3419 (2008).</li><li>Bouchoux, G., Bimbong, R. N. B., Nacer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-Phase+Protonation+Thermochemistry+of+Glutamic+Acid.">Gas-Phase Protonation Thermochemistry of Glutamic Acid.</a> J. Phys. Chem. A. 113 (24), 6666-6676 (2009).</li><li>Zheng, X., Cooks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermochemical+Determinations+by+the+Kinetic+Method+with+Direct+Entropy+Correction.">Thermochemical Determinations by the Kinetic Method with Direct Entropy Correction.</a> J. Phys. Chem. A. 106 (42), 9939-9946 (2002).</li><li>Jones, C. M., Bernier, M., Carson, E., Colyer, K. E., Metz, R., Pawlow, A., Wischow, E. D., Webb, I., Andriole, E. J., Poutsma, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-phase+acidities+of+the+20+protein+amino+acids.">Gas-phase acidities of the 20 protein amino acids.</a> Int. J. Mass Spectrom. 267 (1-3), 54-62 (2007).</li><li>Tan, J. P., Ren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+Gas-Phase+Acidities+of+Cysteine-Polyalanine+Peptides+Using+the+Extended+Kinetic+Method.">Determination of the Gas-Phase Acidities of Cysteine-Polyalanine Peptides Using the Extended Kinetic Method.</a> J. Am. Soc. Mass Spectrom. 18 (2), 188-194 (2007).</li><li>Harrison, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gas-phase+basicities+and+proton+affinities+of+amino+acids+and+peptides.">The gas-phase basicities and proton affinities of amino acids and peptides.</a> Mass Spectrom. Rev. 16 (4), 201-217 (1997).</li><li>Gutte, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Peptides: Synthesis, Structures, and Applications. , (1995).</li><li>Barany, G., Merrifield, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+peptide+synthesis.">Solid-phase peptide synthesis.</a> The Peptides. 2, 1-284 (1979).</li><li>Chan, W. C., White, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fmoc Solid Phase Peptide Synthesis: A Practical Approach. , (2000).</li></ol>

Den framgångsrika mätning av gas-fas surhet en peptid bygger till stor del på valet av lämpliga referensvärden syror. Den idealiska referens syrorna är strukturellt liknande organiska föreningar med väletablerade gas-fas surhetsgrad värden. Referensvärdena syror bör ha liknande strukturer till varandra. Detta kommer att säkerställa en likartad entropi deprotonation för varje referens syrorna i uppsättningen. Referensvärdena syror bör ha syra värden nära de av peptiderna. För kortare cysteininnehålla...

De acidities av aminosyror är bland de viktigaste termokemiska egenskaper som påverkar strukturer, reaktivitet, och den fällbara-utspelas processer proteiner 9,19. Individuella aminosyrarester visar ofta olika effektiva acidities beroende på sina platser i proteiner. I synnerhet resterna belägna vid de aktiva ställena uppvisar ofta störs avsevärt acidities. Ett sådant exempel är cysteinresten som är bosatt i de aktiva ställena i thioredoxin super-familj av enzymer 20,21. Den aktiva cystein är ovanligt surt jämfört med dem i ovikta proteiner 3-5. Det har föreslagits att den spiralformiga konformationen kan ha ett betydande bidrag till den ovanliga surhet. Det finns omfattande experimentella undersökningar i syra-bas-egenskaper av peptider som utförs i lösningar, särskilt i vattenlösningar 2,6-8. Resultaten var ofta kompliceras av lösningsmedelseffekter7. I själva verket är de flesta av de aktiva ställena i proteiner beläget nära det inre region där lösningsmedelseffekter minimeras 9,10. För att förstå inneboende syra-bas-egenskaper av peptider och proteiner, är det viktigt att genomföra studier i ett lösningsmedel-fri miljö. Här presenterar vi en masspektrometri-metod för bestämning av gasfas-surhet. Den metod kallas den förlängda Cooks kinetisk metod. Denna metod har med framgång tillämpas på ett brett spektrum av kemiska system för bestämning av olika termokemiska egenskaper, såsom gasfas-surhet, protonen affinitet, metalljonen affinitet, elektronstrålekanonen affinitet och den joniseringsenergi 11-15, 22-26. Vi har tillämpat denna metod för att bestämma de gas-fas acidities av en serie av oligo cystein-polyalanin och cystein-polyglycin peptiderna 17,18,27. Dessa studier visar att den N-terminala cysteinet peptides är betydligt surare än motsvarande C-terminala sådana. De höga acidities av den tidigare är sannolikt på grund av de spiralformade konformationella effekterna där tiolatanjon starkt stabiliseras genom interaktion med Helix makro-dipol. På grund av den icke-flyktiga och termiskt labila naturen av peptider, är den kinetiska metoden den mest praktiska tillvägagångssätt tillgängliga för närvarande för att producera någorlunda korrekt syra-bas termokemiska mängder av peptider 28. Den allmänna ordningen och ekvationen associerat med den kinetiska metod visas i figur 1. Fastställandet av gas-fas surhet en peptid (AH) börjar med bildandet av en serie av proton-bundna kluster anjoner, [A • H • A i] ¯ (eller [A ¯ • H + • En i ¯] ¯), i jonkällan regionen av masspektrometer, där A ¯ och Aj ¯ är de deprotonerade former av peptiden ochde referensnivåer syror, respektive. De referensnivåer syror är organiska föreningar med kända gas-fas acidities. Referensvärdena syror bör ha strukturer som liknar varandra (men inte nödvändigtvis liknande den av peptiden). Likheten av strukturerna mellan referens syror säkerställer likheten mellan entropies av avprotonering bland dem. Den proton-bundna kluster anjoner är massproducerade väljs och collisionally aktiveras och därefter dissocieras användning kollision-dissociation (CID) experiment för att ge de motsvarande monomera anjoner, A ¯ och Aj ¯, med konstanterna för k och k i, respektive, som visas i fig 1a. Om sekundära splittringarna är försumbara, mängdförhållandet mellan de CID fragmentjoner, [A ¯] / [Aj ¯], representerar ett ungefärligt mått på förhållandet mellan konstanterna, k / k i. Under antagandet att det inte finns någon omvänd aktiveradion hinder för både dissociation kanaler, CID produktjon grenkvoter, ln [A ¯] / [A Jag ¯], kommer linjärt korrelerad till gasfas surhet av peptiden (Δ acid H) och för de referens-syror (Δ acid H I), som visas i figur 1b. I denna ekvation är Δ syra H avg den genomsnittliga gas-fas surhet av referens-syror, är Δ (Δ S) entropin sikt (vilket kan antas vara konstant om referens syrorna är strukturellt lika varandra), R universella gaskonstanten, och T eff är den effektiva temperaturen i systemet. Den effektiva temperaturen är en empirisk parameter som beror på flera experimentella variabler, inklusive kollisionsenergin. Värdet på gasfas-surhetsgrad bestäms genom att konstruera två uppsättningar av termo-kinetiska tomter. Den första uppsättningen är obupprätthålls genom plottning ln ([A ¯] / [Ai ¯]) mot Δ acid H i - Δ syra H avg, såsom visas i fig. 4a. Linjär regression kommer att ge en uppsättning av raka linjer med sluttningarna av X = 1 / RT EFF och fångar i Y = - [Δ acid H - Δ acid H avg] / RT eff - Δ (Δ S) / R. Den andra uppsättningen tomter erhålls genom att plotta de resulterande fångar (Y) från den första uppsättningen mot motsvarande backar (X), som visas i figur 4b. Linjär regression ger en ny linje med en lutning av Δ acid H - Δ acid H avg och en skärningspunkt Δ (Δ S) / R. Värdet av Δ syra H erhålls sedan från lutningen och entropi sikt Δ (Δ S), erhålls fråninterceptet. Experimenten utförs med användning av en trippel kvadrupol masspektrometer gränssnitt till en elektrospray (ESI) jonkälla. Ett schematiskt diagram över masspektrometern visas i figur 2. De CID experiment utförs i massa välja proton-bundna kluster anjoner med den första kvadrupol enheten och låta dem genomgå kollisioner med argonatomer läckte av kollisionen kammare som hålls vid ett tryck av ca 0,5 mTorr. Dissociation produkt joner massa analyseras med den tredje quadrupole enheten. CID-spektra registreras vid flera kollisionsenergier med m / z-intervall tillräckligt bred för att täcka alla möjliga sekundära fragment. CID produkt jonintensiteter mäts genom att ställa instrumentet i den valda reaktion övervakning (SRM) läge där skanningen är inriktad på utvalda produktområden joner. CID experiment utförs vid fyra olika kollisionsenergier, motsvarandecentrum-of-massa energier (E cm) av 1,0, 1,5, 2,0, och 2,5 eV, respektive. I mitten-av-massa energi beräknas enligt följande ekvation: E cm = E lab [m / (M + m)], där E ​​lab är kollisionsenergin i laboratoriet ram, m är massan av argon, och M är massan av den proton-bundna kluster jon. I den här artikeln använder vi oligopeptiden Ala 3 CysNH 2 (A 3 CH) som modell föreningen. C-terminalen är amiderad och tiolgruppen (SH) i cysteinresten blir det sura området. Valet av lämpliga referensvärden syrorna är avgörande för en framgångsrik mätning av gas-fas surhet. Den idealiska referens syrorna är strukturellt likartade (varandra) organiska föreningar med väletablerade gas-fas surhetsgrad värden. Referensvärdena syror bör ha syra värden nära den hos peptiderna. För peptid A 3 CH, halogenerade sex carboxylic syror väljs som referens syror. De sex referenspunkter syror är klorättiksyra (MCAH), bromättiksyra (Mbah), difluorättiksyra (DFAH), diklorättiksyra (DCAH), dibromoacetic syra (DBAH), och tri (TFAH). Två av dem, kommer DFAH och Mbah, användas för att illustrera protokollet.

<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

Aminosyrarester belägna vid olika positioner i veckade proteiner uppvisar ofta olika grader av acidities. Till exempel är en cysteinrest belägen vid eller nära N-terminalen av en helix ofta surare än den vid eller nära C-terminalen 1-6. Även om omfattande experimentella undersökningar i syra-bas-egenskaper hos peptider har utförts i den kondenserade fasen, i synnerhet i vattenlösningar 6-8, är resultaten ofta kompliceras av lösningsmedelseffekter 7. I själva verket är de flesta av de aktiva ställena i proteiner beläget nära det inre region där lösningsmedelseffekter har minimerats 9,10. För att förstå inneboende syra-bas egenskaper hos peptider och proteiner, är det viktigt att utföra studierna i en lösningsmedelsfri miljö. Vi presenterar en metod för att mäta de acidities av oligopeptider i gasfas. Vi använder ett cysteininnehållande oligopeptid, Ala 3 CysNH <sub&gt; 2 (A 3 CH), som modell föreningen. Mätningarna är baserade på väletablerade förlängda Cooks kinetisk metod (figur 1) 11-16. Experimenten utförs med användning av en trippel-kvadrupol masspektrometer gränssnitt med en elektrospray (ESI) jonkälla (figur 2). För varje peptid prov, finns flera referenspunkter syror valt. De referensnivåer syrorna är strukturellt liknande organiska föreningar med kända gas-fas acidities. En lösning av blandningen av peptiden och en referens-syra införes i masspektrometern, och en gas-fas proton-bunden anjonisk kluster av peptid-referens syra bildas. Den proton-bundna kluster massa isoleras och därefter fragmenterad via kollision-dissociation (CID) experiment. De resulterande fragmentjon abundances analyseras med en relation mellan acidities och klustret jon dissociation kinetik. Den gasfas surhet av peptiden är sedan obtaiNed genom linjär regression av termo-kinetiska tomter 17,18. Metoden kan tillämpas på en mängd olika molekylära system, däribland organiska föreningar, aminosyror och deras derivat, oligonukleotider och oligopeptider. Genom att jämföra gas-fas acidities mätt experimentellt med de beräknade värdena för olika konformerer kan konformationella effekter på acidities utvärderas.

<ol><li> CID gafflingsinställningarna experiment ger information om de relativa acidities av peptiden jämfört med de valda referens syror. Två representativa CID spektra av peptiden (A 3 CH) med två referens syror, DFAH och Mbah, visas i figur 3. I fig. 3a jonen överflöd (topphöjd) av peptiden jon är svagare än den för DFA ¯, och i figur 3b, är den jon överflöd av peptiden jon starkare än MBA ¯. De två spektra tyder på att gasfas surhet av peptiden är inom område...

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

Fastställandet av gas-fas acidities av cysteininnehållande oligopeptider beskrivs. Experimenten utförs med användning av en trippel kvadrupol masspektrometer. De relativa acidities av peptiderna mäts med kollisionen-dissociation experiment, och de kvantitativa acidities fastställs med utökade Cooks kinetisk metod.

Fastställande av gasfas Acidities av 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

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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 hög genomströmning Amylos Bestämning i frystorkad potatisknöl Prover

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.

Struktur och samordning Fastställande av peptid-metallkomplex Använda 1D och 2D<sup&gt; 1</sup&gt; H-NMR

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.

Från konstruktioner till kristaller - Mot strukturbestämning av β-fat yttermembranproteiner

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.

Fastställande av karbonylsulfid funktionella grupper i Biooljor genom potentiometrisk titrering: Den 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.

Bestämning av termodynamiska egenskaper av alkaliska jord-flytande metallegeringar med elektromotorisk kraft-tekniken

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.

Förgrymmat höghållfasta sampolymer Aramid fibrer för att möjliggöra bestämning av mekaniska 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.

Syntes och strukturbestämning av µ-Conotoxin PIIIA isomerer med olika 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.

Jon mobilitet-masspektrometri tekniker för att bestämma strukturen och mekanismerna för metall Jon igenkänning och redox aktivitet av metall bindning 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.

Bestämning av fotoisomerisationskvantutbytet för en Hydrazone Photoswitch

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

Analysera bekämpningsmedel i avokado med hjälp av QuEChERS-metoden och 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%.

Förberedelse av avokadoprov med hjälp av QuEChERS-metoden med ammoniumformiat för analys av bekämpningsmedel

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.