Name: synthesis and mass spectrometry analysis of oligo-peptoids
Uploaded: 2018-02-21T13:00:00
Description: Watch this Scientific Journal Video about Synthesis and Mass Spectrometry Analysis of Oligo-peptoids at JoVE.com

Les auteurs aimeraient remercier M. Michael Connolly et Dr Ronald Zuckermann (The Molecular Foundry, Lawrence Berkeley National Laboratory) de soutien technique dans la synthèse de peptoid. Nous reconnaissons l’appui de la National Science Foundation (CHE-1301505). Toutes les expériences de spectrométrie de masse ont été menées à l’installation de spectrométrie de masse de chimie à l’Université du Pacifique.

1. synthèse de la Peptoid
NOTE : La synthèse commence avec l’activation de la résine par un gonflement de la résine et en supprimant le groupe protecteur. Elle est suivie par la chaîne de peptoid sur la résine par le biais de répétition monomère addition cycles de plus en plus. Le premier monomère couplé à la résine est le résidu C-terminal. Le peptoid est allongé de l’extrémité C-terminale à l’extrémité N-terminale. Une fois la séquence peptoid désiré est atteint, ...

<ol>
	<li>Sun, J., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptoid+Polymers:+A+Highly+Designable+Bioinspired+Material.">Peptoid Polymers: A Highly Designable Bioinspired Material.</a> ACS Nano. 7 (6), 4715-4732 (2013).</li><li>Fowler, S. A., Blackwell, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+in+peptoids:+Recent+advances+toward+deciphering+the+structural+requirements+for+biological+function.">Structure-function relationships in peptoids: Recent advances toward deciphering the structural requirements for biological function.</a> Org. Biomol. Chem. 7 (8), 1508-1524 (2009).</li><li>Chongsiriwatana, N. P., Patch, J. A., Czyzewski, A. M., Dohm, M. T., Ivankin, A., Gidalevitz, D., Zuckermann, R. N., Barron, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptoids+that+mimic+the+structure,+function,+and+mechanism+of+helical+antimicrobial+peptides.">Peptoids that mimic the structure, function, and mechanism of helical antimicrobial peptides.</a> Proc. Natl. Acad. Sci. U. S. A. 105 (8), 2794-2799 (2008).</li><li>Kruijtzer, J. A., Nijenhuis, W. A., Wanders, N., Gispen, W. H., Liskamp, R. M., Adan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptoid-Peptide+Hybrids+as+Potent+Novel+Melanocortin+Receptor.">Peptoid-Peptide Hybrids as Potent Novel Melanocortin Receptor.</a> J. Med. Chem. 48 (13), 4224-4230 (2005).</li><li>Liu, B., Alluri, P. 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Soc. 135 (35), 13015-13022 (2013).</li><li>Olivier, G. K., Cho, A., Sanii, B., Connolly, M. D., Tran, H., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-Mimetic+Peptoid+Nanosheets+for+Molecular+Recognition.">Antibody-Mimetic Peptoid Nanosheets for Molecular Recognition.</a> ACS Nano. 7 (10), 9276-9286 (2013).</li><li>Olsen, C. A., Ziegler, H. L., Nielsen, H. M., Frimodt-Moeller, N., Jaroszewski, J. W., Franzyk, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial,+Hemolytic,+and+Cytotoxic+Activities+of+&#946;-Peptoid-Peptide+Hybrid+Oligomers:+Improved+Properties+Compared+to+Natural+AMPs.">Antimicrobial, Hemolytic, and Cytotoxic Activities of &#946;-Peptoid-Peptide Hybrid Oligomers: Improved Properties Compared to Natural AMPs.</a> ChemBioChem. 11 (10), 1356-1360 (2010).</li><li>Lutz, J. -. F., Ouchi, M., Liu, D. R., Sawamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-Controlled+Polymers.">Sequence-Controlled Polymers.</a> Science. 341 (6146), 628 (2013).</li><li>Altuntas, E., Schubert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Polymeromics":+Mass+spectrometry+based+strategies+in+polymer+science+toward+complete+sequencing+approaches:+A+review.">"Polymeromics": Mass spectrometry based strategies in polymer science toward complete sequencing approaches: A review.</a> Anal. Chim. Acta. 808, 56-69 (2014).</li><li>Paulick, M. G., Hart, K. M., Brinner, K. M., Tjandra, M., Charych, D. H., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavable+Hydrophilic+Linker+for+One-Bead-One-Compound+Sequencing+of+Oligomer+Libraries+by+Tandem+Mass+Spectrometry.">Cleavable Hydrophilic Linker for One-Bead-One-Compound Sequencing of Oligomer Libraries by Tandem Mass Spectrometry.</a> J. Comb. Chem. 8 (3), 417-426 (2006).</li><li>Thakkar, A., Cohen, A. S., Connolly, M. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+N-substituted+glycine+peptoid+libraries.">Synthesis of N-substituted glycine peptoid libraries.</a> Methods Enzymol. 267, 437-447 (1996).</li><li>Zuckermann, R. N., Kerr, J. M., Kent, S. B. H., Moos, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+method+for+the+preparation+of+peptoids+[oligo(N-substituted+glycines)]+by+submonomer+solid-phase+synthesis.">Efficient method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase synthesis.</a> J. Am. Chem. Soc. 114 (26), 10646-10647 (1992).</li><li>Utku, Y., Rohatgi, A., Yoo, B., Kirshenbaum, K., Zuckermann, R. N., Pohl, N. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Efficient+Method+for+the+Synthesis+of+Peptoids+with+Mixed+Lysine-type/Arginine-type+Monomers+and+Evaluation+of+Their+Anti-leishmanial+Activity.">An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity.</a> J Vis Exp. (117), (2016).</li><li>Morishetti, K. K., Russell, S. C., Zhao, X., Robinson, D. B., 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=Tandem+mass+spectrometry+studies+of+protonated+and+alkali+metalated+peptoids:+Enhanced+sequence+coverage+by+metal+cation+addition.">Tandem mass spectrometry studies of protonated and alkali metalated peptoids: Enhanced sequence coverage by metal cation addition.</a> Int. J. Mass Spectrom. 308 (1), 98-108 (2011).</li><li>Bogdanov, B., Zhao, X., Robinson, D. B., 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=Electron+Capture+Dissociation+Studies+of+the+Fragmentation+Patterns+of+Doubly+Protonated+and+Mixed+Protonated-Sodiated+Peptoids.">Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids.</a> J. Am. Soc. Mass Spectrom. 25 (7), 1202-1216 (2014).</li><li>Ren, J., Tian, Y., Hossain, E., Connolly, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragmentation+Patterns+and+Mechanisms+of+Singly+and+Doubly+Protonated+Peptoids+Studied+by+Collision+Induced+Dissociation.">Fragmentation Patterns and Mechanisms of Singly and Doubly Protonated Peptoids Studied by Collision Induced Dissociation.</a> J. Am. Soc. Mass Spectrom. 27 (4), 646-661 (2016).</li></ol>

Un peptoid nonamer, Ac-Nae-(Npe-Nme)4-NH2, a été synthétisé en utilisant le protocole présenté. Les appareils de synthèse implique un navire seringue comme réaction en phase solide en polypropylène et un agitateur mécanique. Les navires de la réaction sont commercialement disponibles et peu coûteux. Un agitateur mécanique est un dispositif commun dans les laboratoires de chimie. Avec l’utilisation d’un réacteur de type seringue, solutions peuvent être aspirées et poussées hors d...

Peptoids sont une classe de polymères séquence contrôlée avec des structures de base imitant la structure des peptides. Peptoids peut être synthétisé de diverses amines, qui permet peptoids d’exposer des propriétés hautement accordables1,2. Peptoids ont servi de modèles moléculaires pour la recherche biophysique, considérés comme agents thérapeutiques et conçu en tant que ligands pour protéines3,4,5,6. Peptoids ont été élaborés dans une variété de composés biologiquement actifs, tels que les matériaux antisalissure et anticorps-mimétique, agents antimicrobiens et enzyme inhibiteurs7,8,9. Avec un caractère très ordonné et accordable, peptoids ont aussi été considérés comme un type de stockage d’information moléculaire10. La découverte de ces appels de diverses applications pour le développement de méthodes d’analyse efficaces pour caractériser la séquence et la structure des peptoids. Techniques basées sur la spectrométrie en tandem mass ont montré prometteur comme la méthode de choix pour analyser les propriétés de la séquence de polymères séquence-contrôlée, notamment peptoids11,12,13, 14,15. Cependant, corrélant les patrons de fragmentation peptoid ion résultant des études systématiques des études de spectrométrie de masse et l’information structurale de peptoids sont très limitées.
Peptoids peuvent être facilement synthétisés en utilisant une méthode de la phase solide. La méthode bien développée implique une itération de l’un en deux étapes monomère addition cycle16,17. Dans chaque cycle d’addition, une amine résine liée est acétylée par un acides haloacétiques (généralement l’acide bromoacétique, BMA), et elle est suivie d’une réaction de déplacement avec une amine primaire. Bien que la synthèse automatique des protocoles ont été appliqués systématiquement pour la synthèse de peptoid, les peptoids peuvent être synthétisés manuellement avec d’excellents rendements dans une norme chimie laboratoire16,18,19, 20.
Notre laboratoire a adopté la méthode de synthèse de peptoid manuel et simplifié les appareils utilisés dans les méthodes existantes. Nous avons précédemment étudié les patrons de fragmentation d’une série de peptoids à l’aide de MS/MS techniques21,22,23. Nos résultats montrent que peptoids produire fragmentations caractéristiques lorsqu’elles sont soumises à la dissociation induite par collision (CID)21,23 ou capture d’électrons expériences22 dissociation (DPE). Dans cet article, nous démontrons comment oligo-peptoids peut être synthétisée dans un laboratoire de chimie standard, comment réaliser les expériences de CID à l’aide d’un spectromètre de masse quadripolaire triple et analyser les données spectrales. La peptoid à être synthétisé et caractérisé est un nonamer avec l’acétylation N-terminal et C-terminal amidation, Ac-Nae-(Npe-Nme)4-NH2. La structure de la peptoid est illustrée à la Figure 1.

<table>
	<tbody>
		<tr>
			<td>ESI-triple quadrupole mass spectrometer, Varian 320L</td>
			<td>Agilent Technologies Inc.</td>
			<td></td>
			<td>The mass spectrometer was acquired from Varian, Inc.</td>
		</tr>
		<tr>
			<td>Varian MS workstation, Version 6.9.2, a data acquisition and data review software</td>
			<td>Varian Inc.</td>
			<td></td>
			<td>The software is a part of the Varian 320L package</td>
		</tr>
		<tr>
			<td>Burrell Scientific Wrist-action shaker, Model 75 DD</td>
			<td>Fisher Scientific International Inc.</td>
			<td>14-400-126</td>
			<td></td>
		</tr>
		<tr>
			<td>Hermle Centrifuge, Model Z 206 A</td>
			<td>Hermle Labortechnik GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solid phase reaction vessel, 10 mL</td>
			<td>Torviq</td>
			<td>SF-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure caps for reaction vessels</td>
			<td>Torviq</td>
			<td>PC-SF</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters, pore size 0.2 &#956;m</td>
			<td>Fisher Scientific Inc.</td>
			<td>03-391-3B</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters, pore size 0.45 &#956;m</td>
			<td>Fisher Scientific Inc.</td>
			<td>03-391-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tuges, 50 mL</td>
			<td>VWR International, LLC.</td>
			<td>490001-626</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tuges, 15 mL</td>
			<td>VWR International, LLC.</td>
			<td>490001-620</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemBioDraw, Ultra, Version 12.0</td>
			<td>CambridgeSoft Corporation</td>
			<td></td>
			<td>CambridgeSoft is now part of PerkinElmer Inc.</td>
		</tr>
		<tr>
			<td>Styrofoam cup, 12 Oz</td>
			<td>Common Supermarket</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rink amide resin</td>
			<td>Chem-Impex International, Inc.</td>
			<td>10619</td>
			<td></td>
		</tr>
		<tr>
			<td>Piperidine</td>
			<td>Chem-Impex International, Inc.</td>
			<td>02351</td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>N, N&#8217;-diisopropylcarbodiimide</td>
			<td>Chem-Impex International, Inc.</td>
			<td>00110</td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>Bromoacetic acid</td>
			<td>Chem-Impex International, Inc.</td>
			<td>26843</td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>2-Phenylethylamine</td>
			<td>VWR International, LLC.</td>
			<td>EM8.07334.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Methyoxyethylamine</td>
			<td>Sigma-Aldrich Co. LLC.</td>
			<td>241067</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Boc-ethylenediamine</td>
			<td>VWR International, LLC.</td>
			<td>AAAL19947-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride</td>
			<td>Sigma-Aldrich Co. LLC.</td>
			<td>252845</td>
			<td></td>
		</tr>
		<tr>
			<td>N, N-dimethylformamide</td>
			<td>VWR International, LLC.</td>
			<td>BDH1117-4LG</td>
			<td>Further distillation before use</td>
		</tr>
		<tr>
			<td>N, N-diisopropylethylamine</td>
			<td>Chem-Impex International, Inc.</td>
			<td>00141</td>
			<td></td>
		</tr>
		<tr>
			<td>Triisopropylsilane</td>
			<td>Chem-Impex International, Inc.</td>
			<td>01966</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Chem-Impex International, Inc.</td>
			<td>00289</td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>Millipore MILLI-Q Academic Water Purification System</td>
			<td>Millipore Corporation</td>
			<td>ZMQP60001</td>
			<td>For generating HPLC grade water</td>
		</tr>
		<tr>
			<td>HPLC-grade Water</td>
			<td></td>
			<td></td>
			<td>Produced from Millipore MILLI-Q&#174; Academic Water Purification System</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Pharmco-Aaper</td>
			<td>339USP/NF</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific International, Inc.</td>
			<td>A998-4</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>VWR International, LLC.</td>
			<td>BDH1121-19L</td>
			<td>Further distillation before use</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>VWR International, LLC.</td>
			<td>BDH1113-19L</td>
			<td>Further distillation before use</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>Fresno Oxygen/Barnes Supply</td>
			<td>NIT 50-C-F</td>
			<td>Ultra high purity, 99.9995%</td>
		</tr>
		<tr>
			<td>Argon gas</td>
			<td>Fresno Oxygen/Barnes Supply</td>
			<td>ARG 50-C-F</td>
			<td>Ultra high purity, 99.9995%</td>
		</tr>
	</tbody>
</table>

synthesis and mass spectrometry analysis of oligo-peptoids

Peptoids sont contrôlées par séquence des oligomères de peptide-imitant constitués d’unités de glycine N-alkylés. Parmi les nombreuses applications potentielles, peptoids ont été considérés comme un type de stockage de l’information moléculaire. Analyse par spectrométrie de masse a été considéré la méthode de choix pour les peptoids de séquençage. Peptoids peut être synthétisé par l’intermédiaire de la chimie en phase solide à l’aide d’un cycle répétitif de réaction en deux étapes. Nous présentons ici une méthode pour synthétiser manuellement oligo-peptoids et d’analyser la séquence de la peptoids à l’aide de techniques de spectrométrie de masse (MS/MS) en tandem. L’exemple peptoid est un nonamer composé d’une alternance de N-(2-methyloxyethyl) glycine (Nme) et N-(2-phényléthyle) glycine (Npe), ainsi qu’une glycine N-(2-aminoéthyl) (Nae) à l’extrémité N-terminale. La formule de la séquence de la peptoid est Ac-Nae-(Npe-Nme)4-NH2, Ac désignant le groupe acétyle. La synthèse se déroule dans un réacteur de phase solide disponible dans le commerce. La résine d’amide de patinoire est utilisée comme le support solide pour produire la peptoid avec un groupe amide à l’extrémité C-terminale. Le produit de peptoid qui en résulte est soumis à l’analyse de la séquence à l’aide d’un spectromètre de masse quadripolaire triple couplé à une source d’ionisation par électronébulisation. La mesure de la SM/SM produit une gamme d’ions fragments résultant de la dissociation des peptoid chargée. Les ions fragments sont triées selon les valeurs de leur rapport masse sur charge (m/z). Les valeurs de m/z des ions fragments sont comparées les masses nominales des ions fragments prédites théoriquement, selon le schéma de fragmentation peptoid. L’analyse génère un modèle de fragmentation de la peptoid chargée. Le patron de fragmentation est corrélé à la séquence de monomère de la peptoid neutre. À cet égard, l’analyse MS lit les informations de séquence de la peptoids.

La structure d’un peptoid 9-mer avec l’acétylation N-terminal, Ac-Nae-(Npe-Nme)4-NH2, est illustrée à la Figure 1. Le peptoid a été synthétisé manuellement dans un réacteur en polypropylène fritté via l’approche de la phase solide. Résine d’amide patinoire (0,047 mmol, 84 mg avec chargement 0.56 mmol/g) est utilisé comme le support solide pour produire le peptoid avec une extrémité C-terminale d’amidés. La chaîn...

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Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Synthèse et analyse de spectrométrie de masse des Oligo-peptoids

Un protocole est décrit pour la synthèse manuelle des oligo-peptoids suivie par le séquençage par spectrométrie de masse.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

The overall goal of this procedure, is to demonstrate how to manually synthesize oligo-peptoids and how to analyze their monomer sequence by using mass spectrometry techniques. The method can help beginners to answer key questions in a peptoid field such as how to make peptoids and how to characterize their monomer sequence. The main advantage of this technique is that peptoids with special monomer residues can be synthesized and analyzed in the standard analytical chemistry lab.Even though this method focuses on oligo-peptoids it can also be applied to other systems such as peptides and oligo-nucleotides. Generally individuals new to this technique will struggle because achieving optimal mass spectrometry conditions is difficult without visual demonstration. We first had the idea for this method when we wanted to characterize the structural features of peptoids by tandem mass spectrometry methods.First weigh out 84 milligrams of Rink amide resin and add it to a 10 milliliter polypropylene solid phase reaction vessel. Then insert a plunger into the vessel. Following this, add two milliliters of TMF to the reaction vessel and cap the vessel with a pressure cap.Agitate the vessel on a shaker at room temperature at an angle of movement approximately 12 degrees and 385 oscillations per minute for 30 minutes. After removing the cap, drain the solution to a waste container by pushing the plunger of the reaction vessel. Add two milliliters of 20%piperidine in DMF to the vessel and cap the vessel.After agitating the vessel for two minutes, drain the solution to the waste container. Repeat the procedure, add two milliliters of 20%Pep DMF solution to the vessel and cap the vessel. Agitate on the shaker for 12 minutes and drain the solution to the waste container.After washing the resin, perform a bromo acetylation reaction, by mixing one milliliter of 0.8 molar bromoacetic acid in DMF and one milliliter of 0.8 molar DIC in DMF in a beaker. Transfer the mixture to the reaction vessel containing the resin and cap the vessel. Agitate the vessel on the shaker at room temperature for 20 minutes.Following agitation, drain the solution to the waste container. After washing the resin, perform a displacement reaction but adding one milliliter of one molar 2 methoxyethylamine in DMF to the vessel. After capping the vessel, agitate it at room temperature for 60 minutes.Then drain the solution to the waste container. Once the monomer addition cycles are complete, mix 92 microliters of acetic anhydride, 43.5 microliters of DIPEA, and two milliliters of DMF in a beaker to make the acetylation cocktail. Add the acetylation cocktail to the vessel containing the resin.After capping the vessel, agitate it at room temperature for 60 minutes. When finished, drain the solution to the waste container. After washing the resin with DCM, mix 3.8 milliliters of TFA, 100 microliters of triisopropylsilane and 100 microliters of HPLC grade water in a beaker to make the cleavage cocktail.Add the cleavage cocktail immediately to the vessel containing the resin. After agitating the vessel at room temperature for two hours remove the cap and collect the filtrate solution into a 50 milliliter polypropylene centrifuge tube. Add one milliliter of TFA to the vessel and cap it.After agitating for one minute, collect the filtrate solution into the same centrifuge tube. Next evaporate the TFA with a gentle stream of nitrogen gas until about one milliliter of viscous solution remains. Add 15 milliliters of diethyl ether to the viscous solution and cap the centrifuge tube.Incubate the mixture in a minus 20 degree Celsius freezer for at least two hours to obtain a white solid precipitate. Following incubation, while keeping the centrifuge tube cool pellet the solid using a centrifuge at 4427 times G for 10 minutes. When finished remove the cap of the tube and carefully decant the diethyl ether into a beaker.After washing the solid with diethyl ether dry it with a gentle stream of nitrogen gas. Next, add 10 milliliters of HPLC grade water to dissolve the dried solid. Pass the solution through a nylon syringe filter with a port size of 0.45 micron and collect the filtrate into a pre-weighed 50 milliliter polypropylene centrifuge tube.Shelf freeze the peptoid solution by rotating the centrifuge tube in a 12 ounce double stacked expanded polystyrene cup, containing liquid nitrogen. Then lyophilize the frozen solution overnight to yield the solid peptoid. After preparing the peptoid sample solution for MS analysis remove any possible insoluble particles in the diluted solution, by performing centrifugation at 4427 times G for three minutes.Transfer about 700 microliters of the top portion of the solution into another 1.5 milliliter centrifuge tube to make the peptoid MS working solution with a concentration of about 10 to the minus five molar. Once the MS instrument parameters have been set add approximately 300 microliters of the peptoid MS working solution into a one milliliter syringe. And connect the syringe to the ESI inlet using capillary polyetheretherketone tubing.Then place the syringe under the syringe pump and set the flow rate at 10 microliters per minute to infuse the sample solution into the ESI inlet. Turn on the ESI needle voltage to activate the ESI process and then turn on the detector. Set the display in profile mode and the range of the master charge ratio or M/Z from 100 to 1500.View the profile mass spectrum shown in the profile window. In the method window, use two minutes as the run time. Then open the recording window, fill in a propr file name and start to record the spectrum.Optimize the intensity of the proteinated peptoid peak at M/Z 1265. Set the M/Z range from 1150 to 1350 and adjust the capillary voltage while viewing the peak intensity in millivolts shown in the profile window. Now switch the instrument to the MSMS mode in the method window, use 1265 as the Q1 first mass and leave the Q1 last mass blank.Use 100 as the Q3 first mass and 1400 as the Q3 last mass. Then turn the CID gas on. Following this, set the collision energy at 45 volts.And the collision gas pressure at 1.5 millitorr. View the profile mass spectrum displayed in the profile window observing the peptide ion peak at M/Z 1265 and the peaks with lower M/Z values that represent the fragment ions from the precursor peptoid ion. Now adjust the collision energy and the collision gas pressure to optimize the display of the fragmentation spectrum.In the method window, use two minutes as the runtime. Then open the recording window, fill in a proper file name and start to record the spectrum. The structure of the synthesized nonamer peptoid with n terminal acetylation is shown here.The predicted fragmentation scheme for the peptoid is displayed here, where the proton in the dashed circle indicates the mobile proton that would induce peptoid fragmentation during the CID experiment. If fragmentation occurs at all available amide bonds a total of eight n-terminal fragments and eight C-terminal fragments would form. As an example, the structures and the corresponding master charge values of the B4 ion and the Y5 ion are shown here.The calculated master charge values of all fragment ions from V1 to V8 and from Y1 to Y8 are listed here. The full scan mass spectrum of the proteinated peptoid ion shows a peak at M/Z 1265 corresponding to the proteinated species and a peak at M/Z 1287 corresponding to the sodium ion adapt of the peptoid. The two peaks at M/Z 633 and 644 correspond to doubly proteinated and mixed proteinated sodiated peptoids respectively.The proteinated peptoid MSMS spectrum shows a peak at M/Z 1265 corresponding to the proteinated species and peaks at lower mass to charge values corresponding to the fragment ions from the peptoid ion. Once mastered, the synthesis technique can be done in one to two days and the mass spectrometry technique can be done in one to two hours, if they're performed properly. While attempting the mass spectrometry procedure it is important to check the performance of the instrument.If necessary one may perform an auto tune of the instrument. Following this procedure, other methods like Milk-in modeling can be performed in order to answer additional questions such as what possible confirmations a peptoid could adapt. After its development, this technique paved the way for researchers to explore advanced mass spectrometry technique and structural characterization.After watching this video you should have a good understanding of how to synthesize peptoids manually and how to analyze a monomer sequence by using mass spectrometry techniques. Do not forget that working with chemicals can be extremely hazardous and personal protective equipment should always be used while doing this procedure.

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