Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

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.

1. Preparation of reagents
   NOTE: The liquid reagents are measured using a micropipette and the solid reagents are measured using an analytical balance.
   1. Mix 6.2 mL of N, N'-diisopropylcarbodiimide (DIC) and 43.8 mL of N,N-dimethylformamide (DMF) to prepare a 0.8 M of DIC/DMF solution.
   2. Dissolve 5.56 g of BMA into 50.0 mL of DMF to prepare a 0.8 M BMA/DMF solution.
   3. Mix 2 mL of piperidine (Pip) and 8 mL of DMF to achieve 20% Pip/DMF solution.
   4. Dissolve 1.9 mL of Npe into 13.1 mL of DMF to achieve 1.0 M Npe/DMF.
   5. Dissolve 1.3 mL of Nme into 13.7 mL DMF to achieve 1.0 M Nme/DMF.
   6. Dissolve 0.32 mL 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.
2. Resin activation
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
3. Monomer addition and N-terminal acetylation
   NOTE: Each monomer addition cycle involves two reaction steps, bromoacetylation and displacement.
   1. Carry out the first monomer addition cycle to form Nme-Resin.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. Perform N-terminal acetylation to form Ac-Nae-(Npe-Nme)₄-resin.
   8. Mix 92 µL of acetic anhydride, 43.5 µL of N,N-diisopropylethylamine (DIPEA), and 2 mL of DMF in a beaker to make about 2 mL of acetylation cocktail.
   9. 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.
   10. 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.
   11. 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.
4. Cleavage and purification
   1. Mix 3.8 mL of TFA, 100 µL of triisopropylsilane (TIPS), and 100 µL of HPLC-grade H₂O in a beaker to make 4 mL of cleavage cocktail.
   2. 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.
   3. 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.
   4. Evaporate TFA by blowing in a stream of nitrogen gas gently until about 1 mL viscous solution is left.
   5. Add 15 mL of diethyl ether to the remaining solution, cap the centrifuge tube, and incubate it in a -20 °C freezer for 2 h to overnight. The crude peptoid precipitates as white solid.
   6. 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.
   7. 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.
   8. Dry the solid by gently blowing in a stream of nitrogen gas.
   9. 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 µm, and collect the filtrate into a pre-weighted 50 mL polypropylene centrifuge tube.
   10. 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.
   11. 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.

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.

1. Preparation of sample solution
   1. 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.
   2. Transfer 20 µL 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.
   3. Remove any possible insoluble particles in the diluted sample solution by performing centrifugation at 4,427 x g for 3 min. Transfer about 700 µL 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.
   4. Adjust the concentration of the MS working solution based on the observed signal intensity during mass spectrometry measurements.
   5. An alternative way to remove any possible insoluble particles in the diluted sample solution is to pass the sample solution through a 0.20 µm 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.
2. Recording mass spectra
   1. 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 °C.
   2. Add approximately 300 µL of peptoid MS working solution (about 10^-5 M) into a 500 µL 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 µL/min to infuse the sample solution into the ESI inlet.
   3. 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.
   4. Record the MS spectrum for 2 min. In the "Method Window", use 2 min as the "run time." 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.
   5. 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).
   6. 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 "Method Window," use m/z 1,265 as the "Q1 First Mass" and leave the "Q1 Last Mass" blank. Use m/z 100 as the "Q3 First Mass" and m/z 1,400 as the "Q3 Last Mass."
      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.
   7. Set the collision energy at 40 eV and the collision gas (argon, in this case) pressure at 1.5 mTorr.
   8. 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.
   9. 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.
   10. Record the MS/MS spectrum for 2 min. In the "Method Window," use 2 min as the "run time." Open the recording window and fill in a proper file name, and start to record the spectrum.
   11. Repeat recording 1 – 2 times.
3. 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
   1. 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.
   2. Imagine fragmentation at the 4^th 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.
   3. 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.
   4. Open the recorded MS/MS spectrum of the peptoid using the data review software accompanied with the instrument. Set the Labeling Preferences to "Show Ion Labels" and Label Threshold to 3%. This generates a MS/MS spectrum with m/z values labeled on the peaks.
   5. 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.
   6. 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.