MN-S riconosce il sostegno della National Science Foundation, RUI concedere CHE 1011859. Gli autori ringraziano lo strumento Triade Spettrometria di Massa presso la University of North Carolina a Greensboro per l&#39;uso del spettrometro di massa Thermo Fisher Scientific LTQ Orbitrap XL. Gli autori ringraziano Daniel Todd, Vincenzo Sica, e Brandie Erhmann presso la University of North Carolina a Greensboro per utili suggerimenti e commenti questo lavoro.

Nota: Si prega di consultare tutte le schede di sicurezza pertinenti (MSDS) prima dell&#39;uso. Cloruro di mercurio è una sostanza chimica tossica. Dispositivi di protezione individuale (guanti, occhiali di sicurezza, e camice) devono essere indossati al momento della consegna e tutte le soluzioni collegate. Smaltire le soluzioni in bottiglie rifiuti chimici chiaramente etichettati designate per i metalli pesanti. 1. Preparazione di 5 mm degassificato ammonio formiato tampone, pH 7,5 <ol><li> Sciogliere 0,1576 g di buffer di formiato di ammonio in 450 ml di acqua per HPLC. Regolare il pH della soluzione sopra con 1 M di acido formico e 1 M di idrossido di ammonio a 7,5. Trasferire questa soluzione in un matraccio tarato da 500 ml e aggiungere acqua HPLC per la retta di calibrazione per ottenere una soluzione di ammonio formiato 5 mM. </li><li> Degassare il 5 mM tampone formiato di ammonio sotto un sistema di aspirazione per 10 minuti e spurgo con argon. Ripetere due volte e la soluzione di negozio sotto argon. Il giorno di utilizzo, filtrare la soluzione tampone attraverso un filtro da 0,2 micron before l&#39;uso. </li></ol> 2. Preparazione di mercurio (II) Chloride Soluzioni <ol><li> Pesare 0,2375 g di mercurio (II) cloruro. Sciogliere in 25 ml di 5 mM tampone formiato di ammonio per produrre una soluzione di cloruro di 0,035 M di mercurio (II). </li><li> Aggiungere 0,214 ml di soluzione 0,035 M di mercurio (II) cloruro di 9.785 ml di 5 mM tampone formiato di ammonio per creare una soluzione 7,5 x 10 -4 M. Ricoprire la soluzione con gas argon 7.5 x 10 -4 M mercurio (II). </li></ol> 3. Preparazione del CGGC Stock Solution <ol><li> Sciogliere 2,0 mg di tetrapeptide dicysteinyl, CGGC, in 0,118 ml di acetonitrile grado HPLC e quindi aggiungere 1,0647 ml di 5 formiato di ammonio mM, pH 7,5 tampone che è stato degasata in argon per ottenere una soluzione madre 5 CGGC mm. </li><li> Aggiungere 225 ml di soluzione madre 5 CGGC mM a 1.275 ml di 5 mM formazione ammonio pH 7,5 tampone per dare una soluzione 7,5 x 10 -4 M CGGC. </li></ol> 4. Preparazionedi varie miscele di reazione di mercurio (II) e CGGC <ol><li> Preparazione 1: 0,5 rapporto di mercurio (II): soluzione CGGC <ol><li> Mettere 255 ml di formiato di ammonio 5 mM, pH 7,5 tampone in una provetta da 1,5 ml microcentrifuga. Aggiungere 30 ml di soluzione di mercurio (II) cloruro 7,5 x10 -4 M nel tubo 1,5 ml microcentrifuga con il buffer formiato di ammonio. </li><li> Vortex la soluzione per 10 secondi. Quindi aggiungere 15 ml di 7,5 x 10 -4 soluzione M CGGC nella microcentrifuga 1,5 ml. Vortex la soluzione per 10 secondi. Lasciare riposare la soluzione per 10 minuti prima dell&#39;iniezione nello spettrometro di massa. </li></ol></li><li> Preparazione di rapporto 1: 1 di mercurio (II): soluzione CGGC <ol><li> Mettere 240 ml di formiato di ammonio 5 mM, pH 7,5 tampone in una provetta da 1,5 ml microcentrifuga. Aggiungere 30 ml di soluzione di mercurio (II) cloruro 7,5 x10 -4 M nel tubo 1,5 ml microcentrifuga con il buffer formiato di ammonio. </li><li> Vortex la soluzione per 10 secondi. Poiaggiungere 30 ml di 7,5 x 10 -4 soluzione M CGGC nel tubo 1,5 ml microcentrifuga. Ripetere in un modo simile come descritto nella sezione 4.1. </li></ol></li><li> Preparazione di rapporto 1: 2 di mercurio (II): soluzione CGGC <ol><li> Mettere 210 ml di formiato di ammonio 5 mM, pH 7,5 tampone in una provetta da 1,5 ml microcentrifuga. Aggiungere 30 ml di soluzione di mercurio (II) cloruro 7,5 x10 -4 M nel tubo 1,5 ml microcentrifuga con il buffer formiato di ammonio. </li><li> Vortex la soluzione per 10 secondi. Quindi aggiungere 60 ml di 7,5 x 10 -4 soluzione M CGGC nel tubo 1,5 ml microcentrifuga. Ripetere in un modo simile come descritto nella sezione 4.1. </li></ol></li></ol> 5. Preparazione di PECO Stock Solution <ol><li> Sciogliere 3,5 mg di tetrapeptide dicysteinyl, PECO, in 0,145 ml di acetonitrile grado HPLC per sciogliere il peptide. Poi aggiungere 13.067 ml di 5 mM formiato di ammonio, pH 7,5 tampone che è stata degasata in argon per produrre 0,5 mSoluzione M PECO. </li><li> Agitare la soluzione fino a quando tutti peptide è disciolto. Aggiungere 1,125 ml di soluzione 0,5 mM PECO e 0,375 ml di 5 mM formiato di ammonio, pH 7,5 tampone in una provetta da microcentrifuga da 1,5 ml per ottenere una soluzione 7,5 x 10 -5 M PECO. Vortex fino misti. </li></ol> 6. Preparazione di varie miscele di reazione di mercurio (II) e soluzione PECO <ol><li> Preparazione 1: 0,5 rapporto di mercurio (II): soluzione PECO <ol><li> Mettere 255 ml di formiato di ammonio 5 mM, pH 7,5 tampone in una provetta da 1,5 ml microcentrifuga. Aggiungere 30 ml di soluzione di mercurio (II) cloruro 7,5 x10 -4 M nel tubo 1,5 ml microcentrifuga con il buffer formiato di ammonio. </li><li> Vortex la soluzione per 10 secondi. Quindi aggiungere 15 ml di 7,5 x 10 -4 soluzione M PECO nel tubo 1,5 ml microcentrifuga. Ripetere in un modo simile come descritto nella sezione 4.1. </li></ol></li><li> Preparazione di rapporto 1: 1 di mercurio (II): soluzione PECO <ol><li> Mettere 24081; l di formiato di ammonio 5 mM, pH 7,5 tampone in una provetta da 1,5 ml microcentrifuga. Aggiungere 30 ml di soluzione di mercurio (II) cloruro 7,5 x10 -4 M nel tubo 1,5 ml microcentrifuga con il buffer formiato di ammonio. </li><li> Vortex la soluzione per 10 secondi. Quindi aggiungere 30 ml di 7,5 x 10 -4 soluzione M PECO nel tubo 1,5 ml microcentrifuga. Ripetere in un modo simile come descritto nella sezione 4.1. </li></ol></li><li> Preparazione di rapporto 1: 2 di mercurio (II): soluzione PECO <ol><li> Mettere 210 ml di formiato di ammonio 5 mM, pH 7,5 tampone in una provetta da 1,5 ml microcentrifuga. Aggiungere 30 ml di soluzione di mercurio (II) cloruro 7,5 x10 -4 M nel tubo 1,5 ml microcentrifuga con il buffer formiato di ammonio. </li><li> Vortex la soluzione per 10 secondi. Quindi aggiungere 60 ml di 7,5 x 10 -4 soluzione M PECO nel tubo 1,5 ml microcentrifuga. Ripetere in un modo simile come descritto nella sezione 4.1. </li></ol></li></ol> 7. Analyzing le miscele di reazione di Mercurio (II) e CGGC campioni da Orbitrap ESI Spettrometria di Massa <ol><li> Preparazione del spettrometro di massa ESI 16 <ol><li> Disegnare 100 ml di standard di calibrazione in una siringa di vetro da 500 ml. </li><li> Posizionare la siringa nella culla della siringa della pompa MS, collegare il tubo, e iniettare lo spettrofotometro di massa. </li><li> Impostare il nome del file per la corsa selezionando l&#39;icona del file e digitando il nome del file. </li><li> Selezionare il pulsante dei dati acquisiscono, nel modulo di acquisizione dati e raccogliere 150 scansioni. </li><li> Analizzare il cromatogramma per verificare gli standard di calibrazione aprendo modulo di elaborazione di dati del software. Aprire il modulo, andare al menu File e selezionare &quot;aperto&quot;, e selezionare il file nella finestra di dialogo. Verificare che i picchi nel cromatogramma sono correlati alla massa di addebitare i rapporti degli standard. </li><li> Pulire la siringa di vetro da 500 ml, elaborando 500 microlitri HPLC metanolo e poi dispensare il metanolo in un becher. </li><li> Elaborare 500 ml di metanolo per HPLC nella siringa di vetro e lavare il sistema di cui al punto 7.1.2. </li><li> Selezionare il modulo di impostazione metodo di software per impostare i parametri. Scegliere il menu della modalità di scansione e identificare l&#39;analizzatore come FTMS, e quindi fare clic su &quot;OK&quot;. Poi cliccando sulle varie icone nella pagina vista spettro in tempo reale, impostare i seguenti parametri: Guaina portata del gas: 10, temperatura Fonte: 0, tensione capillare: 37 V, lente del tubo: 95 V, tensione Spray: 4.20 kV , Portata 10.00 ml / min, Analyzer: FTMS, Numero di scansioni: 150. </li></ol></li><li> Esecuzione di campioni CGGC su ESI spettrometro di massa <ol><li> Eseguire il formiato di ammonio pH 7,5 tampone 5 mm. <ol><li> Mettere 500 ml di 5 mM tampone formiato di ammonio nella siringa di vetro da 500 ml, metterlo nella culla della siringa della pompa MS, e collegare il tubo. </li><li> Eseguire tampone attraverso il tubo per 1-2 min. </li><li> Impostare il nome del file per la corsaselezionando l&#39;icona del file e digitando il nome del file. </li><li> Selezionare il pulsante dei dati acquisiscono nel modulo e raccogliere 150 scansioni. </li><li> Fare clic sul pulsante di esecuzione per interrompere la raccolta dopo 150 scansioni vengono raccolti. </li><li> Aprire il modulo del browser di dati, quindi andare al menu File e selezionare &quot;aperto&quot;, e selezionare il file nella finestra di dialogo. Verificare che non picchi a 483, 683, 1.163 e 1.363 m / z sono presenti che assomigliano il peptide o il mercurio (II) -peptide. </li></ol></li><li> Eseguire il 1: 0,5 mercurio (II): soluzione rapporto CGGC. <ol><li> Mettere 250 ml di 1: 0,5 mercurio (II): rapporto CGGC del campione nella siringa. </li><li> Posizionare la siringa nella culla della siringa della pompa MS, collegare il tubo, e il primo l&#39;apparato. </li><li> Selezionare un nome di file per la corsa selezionando l&#39;icona del file e digitando il nome del file. </li><li> Premere il pulsante di dati acquisiscono, nel modulo di acquisizione dati e di raccogliere 150 scansioni e fare clic sul pulsante di esecuzione per fermare la raccolta. </li><li> Aprire il modulo, andare al menu File e selezionare &quot;aperto&quot;, e selezionare il file nella finestra di dialogo. Verificare che il cromatogramma contiene picchi compreso quello per il peptide CGGC sola. </li><li> Lavare la siringa aspirando con 500 microlitri di buffer formiato di ammonio e quindi l&#39;erogazione del buffer formiato di ammonio in un becher. </li><li> Selezionare il pulsante rifiuti MS e lavare la tubazione tre volte con 500 microlitri di buffer formiato di ammonio. </li><li> Lavare la siringa aspirando con 500 microlitri di metanolo e quindi l&#39;erogazione del metanolo in un becher. </li><li> Lavare l&#39;unica volta tubazione da 500 microlitri metanolo. </li><li> Selezionare il pulsante rilevatore carico sui SM. </li><li> Aggiungere 500 microlitri di tampone formiato di ammonio alla siringa. </li><li> Posizionare la siringa nella culla della siringa della pompa MS, collegare il tubo, e il primo l&#39;apparato. </li><li> Selezionare un nome di file per il buffer gestito selezionando l&#39;icona del file e digitando il nome del file. </li><li> stampail pulsante di dati acquisire e raccogliere 150 scansioni e quindi fare clic sul pulsante di esecuzione di arresto. </li><li> Aprire il modulo del browser di dati, andare al menu File e selezionare &quot;aperto&quot;, e selezionare il file nella finestra di dialogo. Verificare che il cromatogramma è privo di picchi del precedente Hg: corsa CGGC. </li></ol></li><li> Eseguire il 1: 1 di mercurio (II): soluzione rapporto CGGC. <ol><li> Mettere 250 ml di 1: 1 di mercurio (II): rapporto CGGC campione nella siringa. </li><li> Ripetere in un modo simile a quello descritto per passaggi 7.2.2.2 a 7.2.2.15. </li></ol></li><li> Eseguire il 1: 2 di mercurio (II): Soluzione rapporto CGGC <ol><li> Mettere 250 ml di 1: 2 di mercurio (II): CGGC campione concentrazione nella siringa. </li><li> Ripetere in un modo simile a quello descritto per passaggi 7.2.2.2 a 7.2.2.15. </li></ol></li></ol></li></ol> 8. Analizzando le miscele di reazione di Mercurio e Campioni PECO di Orbitrap ESI Spettrometria di Massa <ol><li> Esecuzione di campioni PECO su ESI spettrometro di massa <ol><li> Ripetere la procedura di analisi (passaggi 7,1-7,2) utilizzando campioni PECO e miscele di reazione di mercurio (II) e PECO a vari rapporti stechiometrici. </li></ol></li></ol>

<ol>
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United States. , (2012).</li><li>Falcone, G., Foti, C., Gianguzza, A., Giuffr&#232;, O., Napoli, A., Pettignano, A., Piazzese, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequestering+ability+of+some+chelating+agents+towards+methylmercury(II).">Sequestering ability of some chelating agents towards methylmercury(II).</a> Anal. Bioanal. Chem. 405 (2), 881-893 (2013).</li><li>Mah, V., Jalilehvand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+Complex+Formation+with+Mercury(II)+in+Aqueous+Solution+at+Physiological+pH.">Glutathione Complex Formation with Mercury(II) in Aqueous Solution at Physiological pH.</a> Chem. Res. Toxicol. 23, 1815-1823 (2010).</li></ol>

There is no disclosure for the work reported.

Il dicysteinyl tetrapeptide idrofoba CGGC (C 10 H 18 N 4 O 5 S 2; PM = 338) (Figura 1), forma complessi con mercurio (II) come mostrato in Figura 2 e Tabella 1 Inoltre, forma dimeri peptidici e trimeri. incrementale come aumenta la quantità di peptide nella miscela di reazione. Come mostrato dai valori m / z dei dimeri associati [(2M + H) + = 677] e trimeri [(3M + H) + = 1.015...

Current clinical drugs prescribed for chelation therapy of mercury poisoning1 contain thiol group(s), which is/are responsible for binding and sequestering mercury ions2,3. However, studies have shown that these small thiol compounds [dimercaptosuccinic acid (DMSA) and dimercaptopropane-sulfonic acid (DMPS)] are not optimal for mercury chelation therapy4-6. Therefore, there is a need to understand the association and complex formation tendencies of mercury with thiols to enhance the rational drug design of thiol compounds for mercury chelation. Recently, we reported that n-alkyl and aryl dicysteinyl tripeptides with dithiol groups can serve as effective "double anchors" to accommodate the coordination sites of mercury(II) to form 1:1 mercury(II):peptide and 1:2 mercury(II):(peptide)2 complexes7. Additionally, we studied the effect of increasing cysteinyl residues on complex type formations8. In this study, we investigate the association of mercury(II) with two dicysteinyl tetrapeptides, where the cysteinyl residues are separated by two amino acid residues. In order to evaluate the effect of auxiliary binding groups for mercury, the intervening amino acids are either two glycine (unsubstituted) residues or two glutamic acid (gamma-carboxylated) residues. 
The reaction of cysteinyl peptide with mercury(II) requires conditions that will prevent the oxidation of the cysteinyl thiol groups to form disulfide bonds9. Moreover, the association of mercury(II) with cysteinyl peptides to form various types of mercury-peptide complexes is dependent on the initial ratio of mercury(II): peptide in the reaction mixture7,8. The types of mercury-peptide complexes formed in these reaction mixtures can be analyzed by soft-ionization mass spectroscopy, which is a sensitive analytical tool for determining species interactions between metal ions and peptides10-14. Accordingly, it will provide a profile of the various types of mercuriated peptide adducts that are formed under a specified electrospray ionization condition. Here, we will show how cysteinyl peptides and mercury(II) chloride solutions can be prepared in degassed ammonium formate buffer solution blanketed with argon to minimize oxidation. By reacting varying mole equivalents of mercury(II) with dicysteinyl tetrapeptides, we will show how the initial ratio of mercury(II):peptide has an effect on the types of complexes formed. We will also show how electrospray ionization (ESI) mass spectrometry can be used as a characterization tool to provide an accurate stoichiometry of mercury to peptide in the complexes formed. The associated video protocol will demonstrate the experimental conditions for preparing the mercury complexes, the procedure for analyzing the reaction mixtures under specified electrospray ionization conditions, and the characterization of the stoichiometries of mercury(II)-dicysteinyl tetrapeptide complexes, based on the distinct mercury isotope distribution patterns, by using the ChemCal program15. It is intended to assist those who are interested in using ESI orbitrap mass spectrometry to analyze various complexes formed by metal ions that exist in different isotopic forms.

<table><tbody><tr><td>Mercury(II) chloride</td><td>Sigma-Aldrich</td><td>429724</td><td>Highly toxic</td></tr><tr><td>Ammonium formate</td><td>Sigma-Aldrich</td><td>516961</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Fisher</td><td>A512-P500</td><td></td></tr><tr><td>HPLC water</td><td>Fisher</td><td>W5-4</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Fisher</td><td>BP2405-1</td><td></td></tr><tr><td>HPLC Methanol</td><td>Fisher</td><td>A452-4</td><td></td></tr></tbody></table>

a study of the complexation of mercury(ii) with dicysteinyl tetrapeptides by electrospray ionization mass spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

<html> Uno studio è stato effettuato per caratterizzare l&#39;eventuale mercurio peptide complessa composizione per due tetrapeptidi, CGGC e PECO (Figura 1) mediante spettrometria di massa ESI. Complessi di mercurio (II) con CGGC o PECO sono stati studiati facendo reagire le miscele di mercurio (II) e soluzioni di peptidi a tre differenti rapporti molari: 1: 0.5, 1: 1 e 1: 2 (mercurio (II): peptide) . La concentrazione di mercurio (II) era 7,5 x 10 -6 M e la c...

Watch this Scientific Journal Video about A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry at JoVE.com

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

Uno studio della Complessazione di Mercurio (II) con Dicysteinyl Tetrapeptidi da Electrospray ionizzazione Spettrometria di Massa

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

a-study-complexation-mercury-ii-with-dicysteinyl-tetrapeptides

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

7.8K Views.  University of North Carolina at Greensboro. The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

Video: A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Determinazione delle acidità in fase gassosa di oligopeptidi

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

Ricerca

Chimica

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Produzione di disolfuro-stabilizzati peptide transmembrana per gli studi strutturali

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Compact Quantum Dots per singola molecola Imaging

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Ingegneria

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

Analizzando la dinamica delle proteine ​​che utilizzano l&#39;idrogeno Scambio Spettrometria di Massa

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Scambio risolta in tempo ionizzazione elettrospray idrogeno-deuterio Spettrometria di Massa per lo studio delle proteine ​​Struttura e dinamica

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biochimica

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Mass spettrometrica Approcci alla struttura studio e interazioni proteina in liofilizzati Polveri

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

Una piattaforma di spettrometria di massa a scambio idrogeno-deuterio (HDX-MS) per lo studio degli enzimi biosintetici peptidici

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

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

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

Analisi delle molecole complesse e delle loro reazioni sulle superfici mediante mezzi di desorpazione/spettrometria di massa indotta da cluster

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Scambio idrogeno/deuterio basato sull'elettroforesi capillare per la caratterizzazione conformazionale di proteine con spettrometria di massa top-down

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

Uno studio della Complessazione di Mercurio (II) con Dicysteinyl Tetrapeptidi da Electrospray ionizzazione Spettrometria di Massa

Riepilogo

Esplora Altri Video

Capitoli in questo video

Determinazione delle acidità in fase gassosa di oligopeptidi

Produzione di disolfuro-stabilizzati peptide transmembrana per gli studi strutturali

Compact Quantum Dots per singola molecola Imaging

Analizzando la dinamica delle proteine che utilizzano l'idrogeno Scambio Spettrometria di Massa

Scambio risolta in tempo ionizzazione elettrospray idrogeno-deuterio Spettrometria di Massa per lo studio delle proteine Struttura e dinamica

Mass spettrometrica Approcci alla struttura studio e interazioni proteina in liofilizzati Polveri

Una piattaforma di spettrometria di massa a scambio idrogeno-deuterio (HDX-MS) per lo studio degli enzimi biosintetici peptidici

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

Analisi delle molecole complesse e delle loro reazioni sulle superfici mediante mezzi di desorpazione/spettrometria di massa indotta da cluster

Scambio idrogeno/deuterio basato sull'elettroforesi capillare per la caratterizzazione conformazionale di proteine con spettrometria di massa top-down

Uno studio della Complessazione di Mercurio (II) con Dicysteinyl Tetrapeptidi da Electrospray ionizzazione Spettrometria di Massa

Riepilogo

Esplora Altri Video

Capitoli in questo video

Determinazione delle acidità in fase gassosa di oligopeptidi

Produzione di disolfuro-stabilizzati peptide transmembrana per gli studi strutturali

Compact Quantum Dots per singola molecola Imaging

Analizzando la dinamica delle proteine ​​che utilizzano l'idrogeno Scambio Spettrometria di Massa

Scambio risolta in tempo ionizzazione elettrospray idrogeno-deuterio Spettrometria di Massa per lo studio delle proteine ​​Struttura e dinamica

Mass spettrometrica Approcci alla struttura studio e interazioni proteina in liofilizzati Polveri

Una piattaforma di spettrometria di massa a scambio idrogeno-deuterio (HDX-MS) per lo studio degli enzimi biosintetici peptidici

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

Analisi delle molecole complesse e delle loro reazioni sulle superfici mediante mezzi di desorpazione/spettrometria di massa indotta da cluster

Scambio idrogeno/deuterio basato sull'elettroforesi capillare per la caratterizzazione conformazionale di proteine con spettrometria di massa top-down

Analizzando la dinamica delle proteine che utilizzano l'idrogeno Scambio Spettrometria di Massa

Scambio risolta in tempo ionizzazione elettrospray idrogeno-deuterio Spettrometria di Massa per lo studio delle proteine Struttura e dinamica