In-gel Digestion of Proteins for MALDI-TOF

Described here is the in-gel digestion procedure used in the UCSF Mass Spectrometry Facility for generating and recovering peptides from proteins isolated by gel electrophoresis in a manner compatible with subsequent delivery to a MALDI-TOF mass spectrometer.

Notes on the reasoning/opinions behind certain items/actions are displayed in italics.

• 25mM ammonium bicarbonate / 50% Acetonitrile.
• 25mM ammonium bicarbonate pH 8.
• 0.1 ug/ul sequencing grade trypsin (e.g. Promega) in 25mM ammonium bicarbonate pH 8.
• 50% acetonitrile / 5% trifluoroacetic acid.

• Scalpel (or other stainless steel cutting device).
• Vortexer or agitation device for holding 0.5 ml microfuge tubes.
• Siliconized 0.5ml microfuge tubes.
• Gel-loading pipet tips.
• Gel macerating device. Biospec Products (Bartelsville, OK) makes an excellent device (Vortex mixer) that is essentially a battery-powered "swizzle stick" with disposable teflon tips.
• Incubator or water bath capable of maintaining 37 deg C.
• Speed-Vac.

1. Trim away the unstained portions of gel containing bands of interest with scalpel and place the stained gel into siliconized microfuge tubes. Also prepare a sample containing protein-free gel pieces to be carried throughout the process as a negative control.
   Note: If a fast-stain was used, non-stainless steel cutting devices such as razor blades will oxidize and produce final peptide extracts that are rust-colored.
   Note: The siliconized microfuge tubes are used to minimize adsorptive loss of the peptides that will be released from the gel.

2. Macerate the gel pieces (e.g. with the hand-held vortex mixer).
   
   Purpose: to minimize the size of gel particles and faclitate enzyme access to the trapped protein (maceration does this better than dicing the gel with a scalpel).
   Note: Mysticism exists as to the extent of yield improvement resulting from reduced particle size. It is very clear that reduced particle size reduces the necessary solution volume throughout the procedure and thus contributes to reducing salt-inhibiton of MS ionization. It is also clear that decreased particle size increases the chance of clogging HPLC injectors that may be used to separate peptide extracts.

3. Destain and dehydrate the gel particles with ~3 washes (place on mechanical agitator ~10 min.) with a suitable volume (enough to immerse the gel particles) of 25 mM ammonium bicarbonate / 50% acetonitrile. Using gel-loading pipet tips remove and discard the supernatant after each wash. Perform a sufficient number of washes so that the gel pieces shrink, turn an opaque white, and the removed supernatant is colorless.
   Note: The relatively low concentration of 25mM is used to reduce the potential for subsequent salt interference with ionization in the mass spectrometer.
   Note: Gel loading pipet tips have a very narrow aperuture and help prevent withdrawl of acrylamide particles.
   Note: We believe the gel pieces turn white as they become dehydrated and the acrylamide precipitates in the acetonitrile. If the pieces are subsequently left uncovered on a lab bench for several minutes they will become colorless as the acetonitrile evaporates.

4. Dry the destained gel particles for ~30 min in a Speed-Vac.
   Purpose: To maximize the access of enzyme to substrate upon rehydration with enzyme solution. Substantial mysticism exists as to the extent dryness facilitates enzyme access vs. irreversibly precipitating the protein substrate.

5. Rehydrate the gel particles in 25 mM ammonium bicarbonate buffer containing 0.1 ug/ul trypsin. Add only enough solution (can be done in ~5 ul aliquots) so that all is absorbed by the gel.
   Note: the concentration of enzyme results in enzyme:substrate ratios >1:10. Greater ratios are employed than in-solution digestions due to the hindered enzyme access to the gel-trapped protein substrate.
   Note: Mysticism exists as to whether enzyme free in solution results in greater enzyme autolysis.

6. Overlay the gel with 25 mM ammonium bicarbonate buffer pH 8. Add a minimal volume of buffer so that gel pieces remain immersed throughout digestion.

7. Incubate 12-16 hours (overnight) at 37 deg C.
   Note: Substantial mysticism exists as to the extent factors such as digestion time, temperature, presence/absence of agitation, and gel particle size corrrelate to such parameters as the extent of incomplete cleavage, non-specific cleavage, and enzyme autolysis.
   Note: We find incomplete cleavage, non-specific cleavage, and enzyme autolysis with variable degrees of frequency and inconclusive correlation to experimental conditions.

8. Recover peptides by extracting the supernatant, adding additional water first may facilitate this. Perform ~2-3 extractions (place on mechanical agitator ~10 min.) of the digestion mixture with a suitable volume (~twice the volume necessary to immerse gel pieces) of 50% acetonitrile / 5% TFA. Perform a sufficient number of extractions so that the gel pieces shrink and turn an opaque white color. Use gel-loading pipet tips to remove the supernatant after each wash. Save the supernatant (contains the extracted peptides) in siliconized microfuge tubes.
   Note: The siliconized microfuge tubes and high TFA concentration will both minimize adsorptive sample loss.

9. Concentrate the recovered peptides by reducing the final volume of the extracts to < 5 ul in a Speed-Vac and bring back up to 25 ul in 50% acetonitrile / 5% TFA. The amount of volatile salts (ammonium trifluoroacetate and ammonium bicarbonate) can be decreased by a few cycles of adding water and reducing the volume in a Speed-vac.

10. Store the recovered peptides at -20 deg. C until MALDI-TOF is performed.

N.B. This document is over 2 years old and these email addresses may be out of date. D.A. 10/8/98