Microcleavage and Mass Spectrometry Characterization Protocol

About

When synthesizing peptides on resin, especially when performing multiple N-terminus and side-chain reactions on resin, it is helpful to ensure the desired peptide has been properly synthesized within each step of the synthesis. A microcleavage takes a small portion of resin and cleaves the peptide off resin as a sample for mass spectrometry characterization. Based on the predicted molecular weight of the peptide, scientists can determine if the desired peptide is in the sample. The following protocol details these steps.

A PDF version of this protocol is available below:

Reaction Scheme

Glassware and Equipment

• 1 x Adjustable 1000-µL Micropipette
• 1 x 4-mL Dram Vial
• 1 x Vortex Mixer with Tube Foam Insert
• 1 x Fume Hood
• 1 x Air Line
• 1 x 0.20 µm Nylon Filter
• 1 x 1-mL Syringe
• 1 x Mass Spectrometry Vial with Cap
• 1 x Mass Spectrometer (Waters XEVO or Waters SQD2 are preferred)

Materials

The materials needed for this protocol are provided below. The Fisher Scientific catalog numbers are provided in parentheses.

• Trifluoroacetic acid (60-017-61)
• Triisopropylsilane (optional, AC214922500)
• Water (From Milli-Q system or W5-4)

Safety Measures

When performing this protocol, users must wear safety glasses, laboratory gloves, pants, closed-toe shoes, and a fire-retardant laboratory coat. Everything should be performed in an efficient fume hood. The SDS for the involved chemicals are provided below:

CAUTION: TFA is an extremely corrosive liquid; great care must be taken when using this reagent.

Procedures

1. Begin by using a spatula to take a very small portion of resin and add it to a 4-mL dram vial.
2. Use a micropipette to add 300 µL of trifluoroacetic acid (TFA) to the vial, adding it so that the spatula is rinsed of resin.
   • Note: If an initial microcleavage does not show the desired peptide, or if the peptide contains a Cysteine residue, it is helpful to instead add 280 µL of TFA, 10 µL of triisopropylsilane (TIPS), and 10 µL of water.
3. Cap the vial and mix it on the vortex mixer for 30 minutes.
4. After the 30 minutes have passed, open the vial and flow air slowly into the vial to evaporate the TFA.
5. Once all the TFA has been evaporated, add 1 mL of water to the vial using a micropipette.
6. Place the vial back on the vortex mixer and allow it to mix for five minutes.
7. The microcleavage is now complete. Using the 1-mL syringe, draw up the liquid in the dram vial. Attach the 0.2 µm Nylon filter and filter the sample into a mass spectrometry vial. The cap can then be placed on the vial.
   • Note: It helps to flow water through the filter first before filtering the sample.
8. On the computer located near the mass spectrometer, open the MassLynx application and select “Login Samples…”.
   • Note: If running mass spectrometry during high-performance liquid chromatography (HPLC), users can place fractions in a mass spectrometer vial and start at this step.
     • If using the basic column, the HPLC fraction must first be neutralized with acetic acid.
9. In the drop-down menu, select your name.
10. Type in the Job ID, which should consist of advisor_username_date_sample-name.
    • Example: KloxinC_npetrich_20240618_JM22_crude
11. Copy the Job ID and click next. Select whether the run is a Peptide Run or a Protein Run.
12. On the next page, input the number of samples and provide a sample ID, similar to the Job ID. This can be done by pasting in the already copied Job ID and modifying it. Specify the well that the sample will be placed in, as well as the concentration of the sample. Provide a sample ID, well, and concentration for each sample being processed.
13. Open the instrument and place the samples in the specified wells.
14. Close the instrument and select Finish on the computer.
15. Once the run has completed, the integrated peaks can then be compared to predicted peaks of different charges based on the molecular weight of the peptide.
16. For data analysis, select “File”, then “Open Data File” and locate the mass spectra file by locating the D drive > Peptide Data > Click “OK”.
    • The D drive is for the XEVO.
    • The S drive is for the SQD2.
17. Close the integrated window that first appears.
18. Integrate the peak by right clicking and dragging over the range that is to be integrated.
19. The spectra can then be copied and pasted elsewhere to save it.
20. The integrated peak masses at different charges can then be compared to those predicted based off the molecular weight of the peptide. The m/z ratio expected peaks can be calculated for different values of z using the following equation:

21. This procedure can be repeated after other modifications to ensure desired product before performing a full cleavage of peptide from resin.