MALDI-MS analysis

MALDI Protocol for Phosphorylated C-Terminal Domain of RNA polymerase II

Prepare at least 10 µg of protein sample.  More samples might be needed for observing a range of phosphorylation and use the following protocol modified from Pierce’s zip-tip protocol.

1. The protein at ~1mg/ml was digested with 3C protease to remove the GST tag. 3C protease is added to protein solution with a 1:50 molar ratio (3C-protease to GST-CTD) for 3-4 hours at 4 degree. Protein is then diluted with water in serial dilution for MALDI analysis.
2. Adjust protein sample to 0.1% TFA using 10% Trifluoroacetic acid (TFA).
3. Set the pipettor to 10 µL and secure the pipette tip tightly to the end of the pipettor for optimum tip-to-pipettor seal and sample aspiration.
4. Wet tip by aspirating 10 µL of 50% acetonitrile (ACN) in water and then discarding solvent. Repeat thrice.
5. Equilibrate tip by aspirating 10 µL of 0.1% TFA and discarding solvent. Repeat thrice.
6. Aspirate up to 10 µL of the sample (prepared in Step 2) into the C18 tip. For maximum efficiency, dispense and aspirate the sample for 10 cycles.
7. Rinse the tip by aspirating 10 µL of 0.1% TFA/5% ACN (or methanol) and discarding solvent. Repeat once.
8. Slowly aspirate ~2 µL of 0.1% TFA in 50% ACN elution solution and dispense into a clean, empty tube. Cycle the dispensed solution 3-5 times to elute the column in the tip thoroughly.
9. Prepare 40 mg/ml solution of 2,5-Dihydroxybenzoic acid (DHB) matrix based on the company’s recommendations (4 mg of DHB dissolved in 100 µl of 70% ACN in water with 0.1% TFA)
10. Pipette 1 µl of sample onto MALDI plate and 1 µl of the matrix. Mix well without scratching the plate and dry samples by air or by an air blower. Measure MALDI spectra after samples are completely dry. 
11. Alternatively, premixing the protein and matrix before pipetting on the plate is another option. 

Solution: 

1. CTD storage buffer (20mM HEPES pH 7.5, 20mM NaCl)
2. TFA stock solution 10%
3. ACN stock solution 50% and 70%
4. DHB matrix stock solution 40mg/ml

Equipment: AB Voyager-DE PRO MALDI-TOF

Representative Spectra

Reference:

Mayfield JE, Irani S, Escobar EE, Zhang Z, Burkholder NT, Robinson MR, Mehaffey MR, Sipe SN, Yang W, Prescott NA, Kathuria KR, Liu Z, Brodbelt JS, Zhang Y. Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb. Elife. 2019 Aug 6;8. doi: 10.7554/eLife.48725. PubMed PMID: 31385803; PubMed Central PMCID: PMC6715403.

Company protocol for cleanup (Available in the user guide with the product) - https://www.emdmillipore.com/US/en/product/ZipTip-with-0.6-L-C18-resin,MM_NF-ZTC18S096#anchor_PR

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