RapiGest SF Surfactant Protocol


Use the RapiGest SF Surfactant from RapiGest Surfactant from Waters to extract and solubilize hydrophobic proteins and improve in-solution enzymatic digestions of proteins.

RapiGest SF Surfactant is a reagent used to enhance enzymatic digestion of proteins, both in-gel and in-solution. RapiGest SF helps solubilize proteins, making them more susceptible to enzymatic cleavage without inhibiting enzyme activity. Unlike other commonly used denaturants, such as SDS or urea, RapiGest SF does not modify peptides or suppress protease activity. It is compatible with enzymes such as Trypsin, Lys-C, Asp-N, Glu-C, PNGaseF, and other enzymes.



Materials
All solvents should be HPLC grade, NEVER use pipette tips when transferring acids >2% in concentration!
Avoid Contaminations (pdf)

  1. RapiGest SF Powder (Waters Corporation, 5 Pack of 1 ml Vials, 186001860)
  2. 50 mM Ammonium bicarbonate (Fisher, part # A643-500) in H2O (79.1g/mol): 3.955g/100ml)(pH 7.8) make fresh
  3. 500 mM Dithiothreitol DTT (Fisher, part # PI-20291) in H2O
  4. 500 mM Iodoacetamide IAA (Fisher, part # AC12227-0050) in H2O (0.0185g/ml; always prepare fresh, light sensitive)
  5. 500mM HCl
  6. 250 ng / μg/μL Trypsin in 0.01% acetic acid (modified, sequencing grade, Promega, part # V5111, 5 x 20μg/μg)
  7. Water (Fisher, part # W6-4 optima LCMS grade)
  8. Eppendorf LoBind Microcentrifuge Tubes: Protein (Fisher, part # 13-698-794)

Avoid contamination from plasticizers

To avoid contamination from plasticizers, use all fresh solvents. Never use any plastic pipettes to transfer solvents from the original bottles, instead poor the solvent into a beaker. Repeated exposure of solvents to plastics will contaminate the stock solvents with plasticizers. Never use plastic pipettes when handling concentrated (>2%) acids, use glass pipettes or Hamilton syringes (rinse the syringe with water after using it for concentrated acids to avoid corrosion of the metal needle). If you don't know whether your stock solvents already are contaminated, buy new solvents and make sure no one is contaminating them! Keep your solvents in glass bottles, preferably with Teflon lined lids. Never use bottle lids that have foil backed cardboard liners!

Method

  1. Make 0.2% RapiGest diluted in 50 mM Ammonium Bicarbonate pH 7.8 (1 mg RapiGest per 500 μl 50 mM Ammonium Bicarbonate pH 7.8).
  2. Using low adhesion microcentrifuge tubes, add 100 μl 0.2% RapiGest per 100 μl protein mixture (1:1) [final concentration of RapiGest should be 0.1% (w/v)]. If protein is in pellet, add 25-50 μl of 0.1% RapiGest.
  3. Vortex the sample.
  4. Add DTT to a final concentration of 5mM.
  5. Incubate sample at 50oC for 30 minutes.
  6. Cool the sample to room temperature.
  7. Add IAA to a final concentration of 15mM.
  8. Place sample in dark at room temperature for 30 minutes.
  9. Add trypsin for a final concentration of 1:50 enzyme:protein.
    If total amount of protein is very low just add 1-2 μg of trypsin.
  10. Incubate 4h with shaking at 37oC.
  11. Prior to mass spectrometry run, add HCl to a final concentration of 200 mM.
  12. Allow the cleavage reaction to proceed at 37oC for 45 minutes.
  13. A cloudy pellet should appear
  14. Spin sample and separate supernatant from the pellet into a fresh eppendorf tube (e.g. approximately 16,000 x g, for 10 minutes).
  15. Spin again if needed to make sure the cloudy material is completely removed
  16. Also you can add 5% acetonitile or Buffer A (2-5% acetonitrile in water, 0.1% formic acid) to dilute the sample
  17. Proceed with LC-MS analysis of the supernatant.

Resources

  1. RapiGest Surfactant from Waters

References

  1. Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping. Nomura, Eiko, et. al. J. Mass Spect. 2004; 39: 202-207.
  2. Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Yu, Ying-Qing, et. al. Anal. Chem. 75(21): 6023-6028.
  3. A complete peptide mapping of membrane proteins: a novel surfactant aiding the enzymatic digestion of bacteriorhodopsin. Letter to the Editor. Rapid Comm in Mass Spectrom. Yu, Ying-Qing, et. al. 2004; 18; 711-715.
  4. Acid-labile surfactant assists in-solution digestion of proteins resistant to enzymatic attack. Suder, Piotr, et. al. Letter to the Editor. Rapid Comm in Mass Spectrom. 2004; 18; 822-824.
  5. Novel characterization tool for Mab digestion. Genetics Engin News. 2003; 23(17): 37-41.
  6. Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver. Arnold, Randy et. al. J of Proteome Research. 2003; 3, 653-657.
  7. Lauber MA, Yu Ying-Qing, Brousmiche DW, Hua Z, Koza S, Guthrie E, Magnelli P, Taron CH, Fountain KH. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagetn that Facilitates Sensitive Fluorescence and ESI-MS Detection. Anal. Chem. 2015, 87, 5401-5409