Name: Synthesis and Mass Spectrometry Analysis of Oligo-peptoids (Video) | JoVE Journal
Uploaded: 2018-02-21T13:00:00.000+00:00
Duration: 11 min 44 s
Description: Learn manual synthesis of oligo-peptoids and mass spectrometry sequence analysis for biochemistry research. This protocol helps beginners characterize peptoid structures in standard analytical labs.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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>Preparation of reagents 
	NOTE: The liquid reagents are measured using a micropipette and the solid reagents are measured using an analytical balance.
	<ol>
		<li>Mix 6.2 mL of N, N&#39;-diisopropylcarbodiimide (DIC) and 43.8 mL of N,N-dimethylformamide (DMF) to prepare a 0.8 M of DIC/DMF solution.</li>
		<li>Dissolve 5.56 g of BMA into 50.0 mL of DMF to prepare a 0.8 M BMA/DMF solution.</li>
		<li>Mix 2 mL of piperidine (Pip) and 8 mL of DMF to achieve 20% Pip/DMF solution.</li>
		<li>Dissolve 1.9 mL of Npe into 13.1 mL of DMF to achieve 1.0 M Npe/DMF.</li>
		<li>Dissolve 1.3 mL of Nme into 13.7 mL DMF to achieve 1.0 M Nme/DMF.</li>
		<li>Dissolve 0.32 mL&#160;of Nae into 2 mL of DMF to achieve 1.0 M Nae/DMF. 
		Caution: Most of the chemicals used in the synthesis are hazardous. Trifluoroacetic acid (TFA), DIC, Pip, and BMA are hazardous to the skin, eyes, and respiratory tract. DMF and dichloromethane (DCM) are suspected carcinogens. All reactions should be performed in a fume hood and appropriate personal protective equipment should be used. Please check the material safety data sheet (MSDS) of all chemicals used in the synthesis.</li>
	</ol>
	</li>
	<li>Resin activation
	<ol>
		<li>Measure out 84 mg of Rink amide resin (Resin, 0.047 mmol, loading 0.56 mmol/g) and add it to a 10 mL polypropylene solid-phase reaction vessel. Insert the plunger into the vessel.</li>
		<li>Add 2 mL of DMF to the reaction vessel and cap the vessel with a pressure cap. Place the vessel on a shaker, and agitate the vessel at room temperature at an angle of movement of approximately 12 degrees and 385 oscillations/min for 30 min. Drain the solution to a waste container by removing the cap and pushing the plunger of the reaction vessel.</li>
		<li>Add 2 mL of 20% Pip/DMF solution to the vessel and cap the vessel. Agitate it on the shaker for 2 min, and drain the solution to the waste container.</li>
		<li>Add 2 mL of 20% Pip/DMF solution to the vessel, cap the vessel, and agitate it at room temperature for 12 min. Remove the cap and drain the solution to the waste container.</li>
		<li>Wash the resin by adding 1 mL of DMF, capping the vessel, and agitating the vessel for 1 min. Remove the cap and drain the solution by pushing the plunger. Wash the Resin with DMF for 4 additional times.</li>
	</ol>
	</li>
	<li>Monomer addition and N-terminal acetylation 
	NOTE: Each monomer addition cycle involves two reaction steps, bromoacetylation and displacement.
	<ol>
		<li>Carry out the first monomer addition cycle to form Nme-Resin.</li>
		<li>Perform a bromoacetylation reaction. Mix 1 mL of 0.8 M BMA/DMF solution and 1 mL of 0.8 M DIC/DMF solution in a beaker. Transfer the mixture to a reaction vessel containing Resin and cap the vessel. Place the vessel on the shaker and agitate it at room temperature for 20 min. Remove the cap and drain the solution to the waste container.</li>
		<li>Wash the resin by adding 1 mL of DMF, capping the vessel, agitating it for 1 min, and draining the solution by pushing the plunger. Wash the resin by adding 1 mL of DCM, agitating the vessel for 1 min, and draining the solution. Wash the resin with DCM once more, and then wash it with DMF twice.</li>
		<li>Perform a displacement reaction. Add 1 mL of 1.0 M Nme/DMF solution, cap the vessel, agitate the vessel at room temperature for 60 min. Remove the cap and drain the solution by pushing the plunger.</li>
		<li>Wash the resin by adding 1 mL DMF, agitating the vessel for 1 min, and draining the solution by pushing the plunger. Wash the resin by drawing in 1 mL DCM, agitating for 1 min, and draining the solution. Wash DCM once more, followed by washing with DMF twice.</li>
		<li>Repeat monomer addition cycles from steps 1.3.2 to 1.3.5 to form Nae-(Npe-Nme)4-resin. When repeating the step of displacement reaction (1.3.4), use a specific amine solution according to the peptoid sequence. 
		NOTE: The peptoid chain is elongated from the C-terminus to the N-terminus with the C-terminal residue bound to the Resin.</li>
		<li>Perform N-terminal acetylation to form Ac-Nae-(Npe-Nme)4-resin.</li>
		<li>Mix 92 &#181;L of acetic anhydride, 43.5 &#181;L of N,N-diisopropylethylamine (DIPEA), and 2 mL of DMF in a beaker to make about 2 mL of acetylation cocktail.</li>
		<li>Add 2 mL of acetylation cocktail to the vessel containing resin, cap the vessel, and agitate it at room temperature for 60 min. Remove the cap and drain the solution by pushing the plunger.</li>
		<li>Wash the resin by adding 1 mL of DMF, agitating the vessel for 1 min, and draining the solution by pushing the plunger. Wash the resin by adding 1 mL of DCM, agitating the vessel for 1 min, and draining the solution. Repeat washing the resin with DCM once more, then wash with DMF twice more.</li>
		<li>Wash the resin by adding 1 mL of DCM, agitating the vessel for 1 min, and draining the solution by pushing the plunger. Repeat washing with DCM twice. Remove the cap and let the resin air-dry in the reaction vessel for 10 min.</li>
	</ol>
	</li>
	<li>Cleavage and purification
	<ol>
		<li>Mix 3.8 mL of TFA, 100 &#181;L of triisopropylsilane (TIPS), and 100 &#181;L of HPLC-grade H2O in a beaker to make 4 mL of cleavage cocktail.</li>
		<li>Add 4 mL of freshly made cleavage cocktail to the vessel containing resin, cap the vessel, and agitate it at room temperature for 2 h.</li>
		<li>Remove the cap and collect the filtrate solution into a 50 mL polypropylene centrifuge tube. Add 1 mL of TFA to the vessel, cap it, and agitate for 1 min. Collect the filtrate solution into the same centrifuge tube.</li>
		<li>Evaporate TFA by blowing in a stream of nitrogen gas gently until about 1 mL viscous solution is left.</li>
		<li>Add 15 mL of diethyl ether to the remaining solution, cap the centrifuge tube, and incubate it in a -20 &#176;C freezer for 2 h to overnight. The crude peptoid&#160;precipitates as white solid.</li>
		<li>Pellet the solid using a centrifuge at 4,427 x g for 10 min. Remove the cap of the centrifuge tube and decant diethyl ether into a beaker carefully without losing the solid. 
		Caution: Diethyl ether is a flammable organic solvent. Please use a diethyl ether safe centrifuge.</li>
		<li>Wash the solid by adding 10 mL of ice-cold diethyl ether into the centrifuge tube containing the solid, capping the tube, and placing it in the centrifuge. Perform centrifugation at 4,427 x g for 10 min. Remove the centrifuge tube from the centrifuge and decant diethyl ether into a beaker carefully without losing the solid.</li>
		<li>Dry the solid by gently blowing in a stream of nitrogen gas.</li>
		<li>Add 10 mL of HPLC-grade H2O to dissolve the dried solid. Pass the solution through a nylon syringe filter with pore size of 0.45 &#181;m, and collect the filtrate into a pre-weighted 50 mL polypropylene centrifuge tube.</li>
		<li>Shell freeze the solution by placing and rotating the centrifuge tube containing the peptoid solution in a 12-ounce, double-stacked expanded polystyrene cup 1/3 filled with liquid nitrogen. Lyophilize the frozen solution overnight to yield solid peptoid.</li>
		<li>Repeat lyophilization one more time by dissolving the solid peptoid into 10 mL of HPLC-grade H2O, shell freezing in liquid nitrogen, and lyophilizing overnight. The resulting peptoid is sufficiently pure for sequence analysis by mass spectrometry.</li>
	</ol>
	</li>
</ol>
2. MS Measurements and Sequence Analysis
NOTE: The MS/MS experiment is carried out in a triple-quadrupole mass spectrometer coupled to an electrospray ionization (ESI) source. Data collection is controlled by using the data acquisition software accompanied with the instrument. The general procedure includes 1) performing the full scan mass spectrometry experiment and recording the mass spectrum, 2) performing the CID MS/MS experiment and recording the MS/MS spectrum, and 3) comparing the MS/MS spectral data with theoretical fragmentation scheme predicted based on the structural feature of the peptoid.
<ol>
	<li>Preparation of sample solution
	<ol>
		<li>Weigh out 1.0 - 2.0 mg solid peptoid in a 3 - 5 mL glass vial. Add 1 mL mixed solvent of acetonitrile and water (ACN/H2O, 1:1, v/v) to dissolve the peptoid. This gives the stock sample solution with a concentration of about 10-3 M.</li>
		<li>Transfer 20 &#181;L of stock solution into a 1.5 mL centrifuge tube and add 1 mL mixed solvent of ACN/H2O to yield a diluted sample solution of about 10-5 M.</li>
		<li>Remove any possible insoluble particles in the diluted sample solution by performing centrifugation at 4,427 x g for 3 min. Transfer about 700 &#181;L of the top portion of the solution into another 1.5 mL centrifuge tube to make the peptoid MS working solution with concentration of about 10-5 M.</li>
		<li>Adjust the concentration of the MS working solution based on the observed signal intensity during mass spectrometry measurements.</li>
		<li>An alternative way to remove any possible insoluble particles in the diluted sample solution is to pass the sample solution through a 0.20 &#181;m syringe filter and collect the filtrate into a 1.5 mL centrifuge tube to make the peptoid MS working solution of about 10-5 M.</li>
	</ol>
	</li>
	<li>Recording mass spectra
	<ol>
		<li>Run mixed solvent of ACN/H2O (1:1, v/v) through the electrospray ionization (ESI) source and set up the instrument in a standard positive ion, full scan mass spectrometry mode with a mass-to-charge ratio (m/z) range of 100 - 1500. 
		Note: 
		Typical operating parameters: 
		ESI needle voltage, 5 kV 
		Capillary voltage, 40 V 
		Drying gas (nitrogen gas) temperature, 200 &#176;C.</li>
		<li>Add approximately 300 &#181;L of peptoid MS working solution (about 10-5 M) into a 500 &#181;L or a 1 mL syringe and connect the syringe to the ESI inlet using capillary polyether ether ketone (PEEK) tubing. Place the syringe onto the syringe pump and set the flow rate at 10 &#181;L/min to infuse the sample solution into the ESI inlet.</li>
		<li>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 m/z range of 100 - 1,500. View a mass spectrum profile shown in the profile window. The peak at m/z 1,265 (displayed as m/z of 1,264.6) corresponds to the peptoid ion or protonated peptoid.</li>
		<li>Record the MS spectrum for 2 min. In the &#34;Method Window&#34;, use 2 min as the &#34;run time.&#34;&#160;Open the recording window and fill in a proper file name, and start to record the spectrum. 
		NOTE: The resulting mass spectrum is shown in Figure 3.</li>
		<li>Optimize the intensity of the peak at m/z of 1,265. Set the m/z range to 1,150 - 1,350 and adjust the capillary voltage while viewing the peak intensity in mV shown in the profile window. For example, increase the capillary voltage to 50 V or higher and view the change of the peak intensity. The optimal peak intensity is around 150 - 200 mV. 
		NOTE: Adjusting the capillary voltage can significantly change the abundance of certain ions. Increasing the capillary voltage may enhance the intensity of the peptoid ion. However, a high capillary voltage may also induce dissociation of the peptoid ion in the ion source, and decrease the observed intensity. Adjusting the temperature of drying gas may enhance the intensity of the peak at m/z 1,265, but it is less effective than adjusting the capillary voltage. If the peak at m/z 1,265 does not reach the optimal intensity, adjust the sample concentration (or example, double the concentration of the MS working solution).</li>
		<li>Switch the instrument to the MS/MS mode. Set the precursor ion at m/z 1,265 and the MS/MS mass range at m/z of 100 - 1,400. In the &#34;Method Window,&#34;&#160;use m/z 1,265 as the &#34;Q1 First Mass&#34; and leave the &#34;Q1 Last Mass&#34; blank. Use m/z 100 as the &#34;Q3 First Mass&#34; and m/z 1,400 as the &#34;Q3 Last Mass.&#34; 
		NOTE: In the MS/MS mode, the first quadrupole unit (Q1) functions as a mass filter to isolate the peptoid ion as the precursor ion, the second quadrupole unit (Q2) is a collision cell, and the third quadrupole unit (Q3) functions as a mass analyzer.</li>
		<li>Set the collision energy at 40 eV and the collision gas (argon, in this case) pressure at 1.5 mTorr.</li>
		<li>View a mass spectrum profile displayed in the profile window. The peak at m/z of 1,265 corresponds to the peptoid ion, and the peaks with lower m/z values represent the fragments from the peptoid ion.</li>
		<li>Adjust the collision energy to optimize the display of the fragmentation spectrum. For example, increase the collision energy to 45 eV and view the change of the spectrum profile. 
		NOTE: In general, increasing the collision energy will enhance the abundance of the fragment ions and reduce the abundance of the peptoid ion. Increasing the collision gas pressure will also enhance the abundance of the fragment ions. 
		Caution: Do not increase the collision gas pressure beyond 2 mTorr.</li>
		<li>Record the MS/MS spectrum for 2 min. In the &#34;Method Window,&#34;&#160;use 2 min as the &#34;run time.&#34;&#160;Open the recording window and fill in a proper file name, and start to record the spectrum.</li>
		<li>Repeat recording 1 - 2 times.</li>
	</ol>
	</li>
	<li>Peptoid sequence analysis 
	NOTE: Under the CID condition, the peptoid ion would fragment at the amide bonds along the peptoid backbone to produce a series of N-terminal fragments called B-ions, and a series of C-terminal fragments called Y-ions
	<ol>
		<li>Draw out the chemical structure of the acetylated peptoid by placing the N-terminus on the left side and the C-terminus on the right side, and draw a dashed line at each amide bond to generate a fragmentation scheme as shown in Figure 2a. Draw a proton with a dashed circle to indicate the charge carrier. Starting from the left side of the structure, place labels on the dashed lines to indicate the N-terminal fragments as B1, B2, to B8. Starting from the right side, place labels on the dashed lines to indicate the C-terminal fragments as Y1, Y2, to Y8. 
		NOTE: A chemical drawing software can be utilized to draw the peptoid structure.</li>
		<li>Imagine fragmentation at the 4th amide bond from the left side, and draw the structures of the N-terminal fragment and the C-terminal fragment with proper formal charges, as shown in Figure 2b. Place the label of B4 on the N-terminal fragment, and place the label of Y5 on the C-terminal fragment. Calculate the m/z value of B4 by summing up the nominal masses of the elements in the structure to yield a value of 580, and place m/z 580 on the N-terminal fragment. Calculate the m/z value of Y5 and place m/z 685 on the C-terminal fragment.</li>
		<li>Calculate the m/z values for all eight B ions and all eight Y ions, and assemble them in a table, as shown in Table 1. 
		NOTE: The m/z values of the fragments can be calculated using a chemical drawing software.</li>
		<li>Open the recorded MS/MS spectrum of the peptoid using the data review software accompanied with the instrument. Set the Labeling Preferences to &#34;Show Ion Labels&#34; and Label Threshold to 3%. This generates a MS/MS spectrum with m/z values labeled on the peaks.</li>
		<li>Export the MS/MS spectral data as a text file in the CSV format and reconstruct the spectrum using data processing software. Place m/z values on the peaks. The resulting MS/MS spectrum is shown in Figure 4. 
		NOTE: Some mass spectrometers are equipped with data processing software capable for generating the MS/MS spectrum. In this case, exporting the MS/MS spectral data is not necessary.</li>
		<li>Assign the m/z values shown on the MS/MS spectrum to those shown in Table 1 to identify the corresponding B- and Y-ions. For example, the peak at m/z 580 is identified as the B4-ion and m/z 685 is identified as the Y5-ion. Label the peaks with corresponding B and Y symbols on the spectrum, as shown in Figure 4.</li>
	</ol>
	</li>
</ol>

<ol><li>Sun, J., Zuckermann, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptoid+Polymers%3A+A+Highly+Designable+Bioinspired+Material%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">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/pubmed?term=((Structure-function+relationships+in+peptoids%3A+Recent+advances+toward+deciphering+the+structural+requirements+for+biological+function%5BTitle%5D)+AND+%22Org.+Biomol.+Chem%22%5BJournal%5D)">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/pubmed?term=((Peptoids+that+mimic+the+structure%2C+function%2C+and+mechanism+of+helical+antimicrobial+peptides%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">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/pubmed?term=((Peptoid-Peptide+Hybrids+as+Potent+Novel+Melanocortin+Receptor%5BTitle%5D)+AND+%22J.+Med.+Chem%22%5BJournal%5D)">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/pubmed?term=((A+Potent+Transactivation+Domain+Mimic+with+Activity+in+Living+Cells%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">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/pubmed?term=((Helical+Peptoid+Mimics+of+Magainin-2+Amide%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">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/pubmed?term=((Antifouling+Glycocalyx-Mimetic+Peptoids%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">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/pubmed?term=((Antibody-Mimetic+Peptoid+Nanosheets+for+Molecular+Recognition%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">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/pubmed?term=((Antimicrobial%2C+Hemolytic%2C+and+Cytotoxic+Activities+of+%CE%B2-Peptoid-Peptide+Hybrid+Oligomers%3A+Improved+Properties+Compared+to+Natural+AMPs%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">Antimicrobial, Hemolytic, and Cytotoxic Activities of β-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://science.sciencemag.org/content/341/6146/1238149.full">Sequence-Controlled Polymers.</a> Science. 341 (6146), Washington, DC. 628(2013).</li>
<li>Altuntas, E., Schubert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%22Polymeromics%22%3A+Mass+spectrometry+based+strategies+in+polymer+science+toward+complete+sequencing+approaches%3A+A+review%5BTitle%5D)+AND+%22Anal.+Chim.+Acta%22%5BJournal%5D)">"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/pubmed?term=((Cleavable+Hydrophilic+Linker+for+One-Bead-One-Compound+Sequencing+of+Oligomer+Libraries+by+Tandem+Mass+Spectrometry%5BTitle%5D)+AND+%22J.+Comb.+Chem%22%5BJournal%5D)">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/pubmed?term=((High-Throughput+Sequencing+of+Peptoids+and+Peptide-Peptoid+Hybrids+by+Partial+Edman+Degradation+and+Mass+Spectrometry%5BTitle%5D)+AND+%22J.+Comb.+Chem%22%5BJournal%5D)">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/pubmed?term=((Submonomer+Synthesis+of+A+Hybrid+Peptoid-Azapeptoid+Library%5BTitle%5D)+AND+%22ACS+Comb+Sci%22%5BJournal%5D)">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://pubs.acs.org/doi/abs/10.1021/ma200542p">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/pubmed?term=((Synthesis+of+N-substituted+glycine+peptoid+libraries%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">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://pubs.acs.org/doi/abs/10.1021/ja00052a076">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://pubs.acs.org/doi/abs/10.1021/ed100202f">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/pubmed?term=((Solid-phase+submonomer+synthesis+of+peptoid+Polymers+and+their+self-assembly+into+highly-ordered+nanosheets%5BTitle%5D)+AND+%22J.+Visualized+Exp%22%5BJournal%5D)">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/pubmed?term=((An+Efficient+Method+for+the+Synthesis+of+Peptoids+with+Mixed+Lysine-type%2FArginine-type+Monomers+and+Evaluation+of+Their+Anti-leishmanial+Activity%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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.sciencedirect.com/science/article/pii/S1387380611003071">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/pubmed?term=((Electron+Capture+Dissociation+Studies+of+the+Fragmentation+Patterns+of+Doubly+Protonated+and+Mixed+Protonated-Sodiated+Peptoids%5BTitle%5D)+AND+%22J.+Am.+Soc.+Mass+Spectrom%22%5BJournal%5D)">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/pubmed?term=((Fragmentation+Patterns+and+Mechanisms+of+Singly+and+Doubly+Protonated+Peptoids+Studied+by+Collision+Induced+Dissociation%5BTitle%5D)+AND+%22J.+Am.+Soc.+Mass+Spectrom%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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 &amp;mu;m</td><td>Fisher Scientific Inc.</td><td>03-391-3B</td><td></td></tr><tr><td>Syringe filters, pore size 0.45 &amp;mu;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&amp;rsquo;-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&amp;reg; 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

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...

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

Research

JoVE Journal

Chemistry

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

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 Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2'-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Oligonucleotide 5′-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5′-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

Chemical Triphosphorylation of Oligonucleotides

Learn manual synthesis of oligo-peptoids and mass spectrometry sequence analysis for biochemistry research. This protocol helps beginners characterize peptoid structures in standard analytical labs.

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Explore More Videos