The authors would like to thank Mr. Michael Connolly and Dr. Ronald Zuckermann (The Molecular Foundry, Lawrence Berkeley National Laboratory) for technique support in peptoid synthesis. We acknowledge the support from the National Science Foundation (CHE-1301505). All mass spectrometry experiments were conducted at the Chemistry Mass Spectrometry Facility at the University of the Pacific.

1. Synthesis of Peptoid
NOTE: The synthesis begins with activating the resin by swelling the resin and removing the protecting group. This is followed by growing the peptoid chain onto the resin through repeating monomer addition cycles. The first monomer coupled to the resin is the C-terminal residue. The peptoid is elongated from the C-terminus to the N-terminus. Once the desired peptoid sequence is achieved, the resin is cleaved off and the peptoid product is purified.
<ol>
	<li>P...

<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. G., Yu, P., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Potent+Transactivation+Domain+Mimic+with+Activity+in+Living+Cells.">A Potent Transactivation Domain Mimic with Activity in Living Cells.</a> J. Am. Chem. Soc. 127 (23), 8254-8255 (2005).</li><li>Patch, J. A., 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=Helical+Peptoid+Mimics+of+Magainin-2+Amide.">Helical Peptoid Mimics of Magainin-2 Amide.</a> J. Am. Chem. Soc. 125 (40), 12092-12093 (2003).</li><li>Ham, H. O., Park, S. H., Kurutz, J. W., Szleifer, I. G., Messersmith, 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=Antifouling+Glycocalyx-Mimetic+Peptoids.">Antifouling Glycocalyx-Mimetic Peptoids.</a> J. Am. Chem. 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. D., Zuckermann, R. N., Pei, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Sequencing+of+Peptoids+and+Peptide-Peptoid+Hybrids+by+Partial+Edman+Degradation+and+Mass+Spectrometry.">High-Throughput Sequencing of Peptoids and Peptide-Peptoid Hybrids by Partial Edman Degradation and Mass Spectrometry.</a> J. Comb. Chem. 11 (2), 294-302 (2009).</li><li>Sarma, B. K., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submonomer+Synthesis+of+A+Hybrid+Peptoid-Azapeptoid+Library.">Submonomer Synthesis of A Hybrid Peptoid-Azapeptoid Library.</a> ACS Comb Sci. 14 (10), 558-564 (2012).</li><li>Li, X., Guo, L., Casiano-Maldonado, M., Zhang, D., 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=Top-Down+Multidimensional+Mass+Spectrometry+Methods+for+Synthetic+Polymer+Analysis.">Top-Down Multidimensional Mass Spectrometry Methods for Synthetic Polymer Analysis.</a> Macromolecules. 44 (12), 4555-4564 (2011).</li><li>Figliozzi, G. M., Goldsmith, R., Ng, S. C., Banville, S. C., 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=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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Multistep+Synthesis+of+a+Bioactive+Peptidomimetic+Oligomer+for+the+Undergraduate+Laboratory.">Rapid Multistep Synthesis of a Bioactive Peptidomimetic Oligomer for the Undergraduate Laboratory.</a> J. Chem. Educ. 87 (6), 637-639 (2010).</li><li>Tran, H., Gael, S. L., Connolly, M. D., 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=Solid-phase+submonomer+synthesis+of+peptoid+Polymers+and+their+self-assembly+into+highly-ordered+nanosheets.">Solid-phase submonomer synthesis of peptoid Polymers and their self-assembly into highly-ordered nanosheets.</a> J. Visualized Exp. (57), e3373 (2011).</li><li>Bolt, H. L., Cobb, S. L., Denny, P. 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>

A nonamer peptoid, Ac-Nae-(Npe-Nme)4-NH2, has been synthesized using the protocol presented. The synthesis apparatus involves a syringe-like polypropylene solid-phase reaction vessel and a mechanical shaker. The reaction vessels are commercially available and low cost. A mechanical shaker is a common apparatus in chemistry laboratories. With the use of a syringe-like reaction vessel, solutions can be drawn into and pushed out of the vessel by manually moving the plunger. This technique allows the mo...

Peptoids are a class of sequence-controlled polymers with backbone structures mimicking the structure of peptides. Peptoids can be synthesized from diverse amines, which enables peptoids to exhibit highly tunable properties1,2. Peptoids have been used as molecular models for biophysical research, considered as therapeutic agents, and designed as ligands for proteins3,4,5,6. Peptoids have been developed into a variety of biologically active compounds, such as anti-fouling and antibody-mimetic materials, antimicrobial agents, and enzyme inhibitors7,8,9. With a highly ordered and tunable nature, peptoids have also been thought of as a type of molecular information storage10. The discovery of these diverse applications calls for the development of efficient analytical methods to characterize the sequence and structure of peptoids. Tandem mass spectrometry-based techniques have shown promise as the method of choice for analyzing the sequence properties of sequence-controlled polymers, including peptoids11,12,13,14,15. However, systematic studies correlating the peptoid ion fragmentation patterns resulting from mass spectrometry studies and the structural information of peptoids are very limited.
Peptoids can be readily synthesized using a solid phase method. The well-developed method involves an iteration of a two-step monomer addition cycle16,17. In each addition cycle, a resin-bound amine is acetylated by a haloacetic acid (typically bromoacetic acid, BMA), and this is followed by a displacement reaction with a primary amine. Although automated synthesis protocols have been routinely applied for peptoid synthesis, peptoids can be synthesized manually with excellent yields in a standard chemistry laboratory16,18,19,20.
Our lab has adopted the method of manual peptoid synthesis and simplified the apparatus used in the existing methods. We have previously studied the fragmentation patterns of a series of peptoids using MS/MS techniques21,22,23. Our results show that peptoids produce characteristic fragmentations when they are subjected to collision-induced dissociation (CID)21,23 or electron-capture dissociation (ECD)22 experiments. In this article, we demonstrate how oligo-peptoids can be synthesized in a standard&#160;chemistry laboratory, how to perform the CID experiments using a triple-quadrupole mass spectrometer, and how to analyze the spectral data. The peptoid to be synthesized and characterized is a nonamer with N-terminal acetylation and C-terminal amidation, Ac-Nae-(Npe-Nme)4-NH2. The structure of the peptoid is shown in 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 are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

The structure of a 9-mer peptoid with N-terminal acetylation, Ac-Nae-(Npe-Nme)4-NH2, is shown in Figure 1. The peptoid was synthesized manually in a fritted polypropylene reaction vessel via solid phase approach. Rink amide resin (0.047 mmol, 84 mg with loading 0.56 mmol/g) is used as the solid support to yield the peptoid with an amidated C-terminus. The peptoid chain is built by multiple cycles of monomer addition. Each monomer additio...

Watch this Scientific Journal Video about Synthesis and Mass Spectrometry Analysis of Oligo-peptoids at JoVE.com

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

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.

synthesis-and-mass-spectrometry-analysis-of-oligo-peptoids

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JoVE Journal

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10.5K ビュー.  University of the Pacific. 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 t...

ビデオ: Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

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.

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

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes which have to be handled using inert conditions, such as the Schlenk technique.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

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.

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.

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

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. &#946;-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of ...