Solution Digestion Protocols (Trypsin)

This recipe is just one example of satisfactory protocols, this one was done primarily for digesting ~100 nMoles of protein in a final volume of trypsin digestion reaction of 100 µl (protein solution about 10% of the volume, i.e., enough to keep cations like Na+ concentration only about 5 mM [above 40-50 mM concentration Na+ or K+ can inhibit subsequent mass spec]). (See added notes in ALL CAPS for doing larger protein quantities for subsequent iTRAQ labeling)

If doing without reduction/alkylation of cysteines, this 100 µl final volume contains:

  • 50 mM NH4HCO3, pH 8 (If DIGESTION IS FOR SUBSEQUENT iTRAQ LABELING, use TRIETHYLAMMONIUM BICARBONATE, but note that iTRAQ digests SHOULD be alkylated, see iTRAQ SAMPLE PREP PROTOCOL)
  • 10% v/v acetonitrile (10 µl)
  • 0.1 µg Promega Gold or Sequencing grade modified (methylated) Trypsin (TO DIGEST 100 ug protein for iTRAQ, use 5.0 ug Trypsin)
  • Incubate at least 3 hours at 48°C (alternately, 16-18 hours at 37°C)
  • (Optional - Stop reaction by addition of 4 µl of Glacial Acetic Acid).
  • SpeedVac to dry down reaction completely to evaporate off NH4HCO3 and acetonitrile. Resuspend in 200 µl H2O with vortexing.
  • Repeat drying down 3X total, but last time dry down to leave ~9-10 µl instead of complete evaporation. (SEE iTRAQ SAMPLE PREP PROTOCOL for DIFFERENT INSTRUCTIONS for iTRAQ)
  • Add 1/9th volume of 1.0% trifluoroacetic acid (TFA) for samples that will be analyzed by MALDI ; OR 1.0% formic acid for samples that will be analyzed by ESI (Electrospray or Nanospray) instruments such as our ABSciex 5600 TripleTOF or QTrap 4000 or Waters Synapt HDMS to make final 0.1% TFA or formic acid concentration (can be done in MS facility).


If doing the reactions with prior reduction/alkylation (which can improve digestion efficiency by preventing disulfide bonds formation, which in turn helps the protein to be fully unfolded and therefore having all tryptic sites exposed):

  • If your protein isn’t already in at least 2.5 mM DTT, add DTT or TCEP to your solution to 2.5 mM and incubate at 50°C for at least 15 min.
  • Then, in a final volume of 20 µl:
    • ~100 nmole (~4 µg) of protein in no more than 10 µl, to keep the DTT concentration down to avoid quenching the iodoacetamide reaction)
    • 50 mM NH4HCO3, pH 8
    • 10 mM iodoacetamide (e.g., add 0.8 µl of 250 mM iodoacetamide)
  • Incubate in the dark for 30 mins at 37°C.
    • After 30 mins, you can quench the reaction somewhat (to avoid subsequent alkylation of the trypsin) by adding an additional 2.5 µl of 100 mM DTT and incubating for another 15-30 mins at 37° (this would make the DTT concentration in your 20+2.5 µl volume at least 11 mM, theoretically enough to stoichiometrically titrate all the 10 mM original concentration of iodoacetamide); however, we have no direct evidence that this is necessary and have gotten very nice trypsin digestions without this quenching.
  • Bring the volume of the protein alkylation reaction up to a final volume of 100 µl containing:
    • 50 mM NH4HCO3, pH 8
    • 10% v/v acetonitrile (10 µl)
    • 0.1 µg Promega Gold or Sequencing grade modified (methylated) Trypsin (basic rule of thumb is 1 µg trypsin for every 20-100 µg of protein)
  • Incubate >3 hours at 48°C (alternately, 16-18 hours at 37°C)
  • (Optional - Stop reaction by addition of 4 µl of Glacial Acetic Acid).
  • Dry down reaction completely to evaporate off NH4HCO3, and acetonitrile. (Lyophilization is better than SpeedVac if you have the option, but either works). Resuspend in 200 µl H2O with vortexing.
  • Repeat drying down 3X total, but last time dry down to leave ~10 µl instead of complete evaporation.
  • Add 1/9th volume of [1.0% trifluoroacetic acid (TFA) for samples that will be analyzed by MALDI ; OR 1.0% formic acid for samples that will be analyzed by ESI (Electrospray or Nanaospray ) instruments such as our ABSciex QTrap 4000 or Waters Synapt HDMS] to make final 0.1% TFA or Formic acid concentration (can be done in MS facility)

Although we have not tested this in-house, there is also evidence that adding the acid-labile surfactant sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxyl]-1-propanesulfonate, marketed by Waters under the name RapiGest (p/n 186001860, 186001861, 186002123, 186002122) to digests will produce more complete digests, particularly in the case of more difficult proteins (see for example Rapid Commun. Mass Spectrom. 2004; 18: 822–824). There is also evidence that 5% trifluroethanol (TFE) may act as an excellent denaturant for digestion and subsequent mass spectrometrometry, replacing the 10% acetonitrile or other organics used in some protocols, and the urea used in others.

Note that multiple other protocols exist, all of which probably work fine for most digests, with only occasional individual proteins benefiting from one protocol vs. another.