Proteins & Mass Spectrometry

Mass spectrometry and tandem MS/MS

Protocol from Current Protocols in Protein Science

In-gel Digestion Protocol

• Stained polyacrylamide gel containing protein band of interest
• 25 mM ammonium bicarbonate/50% (v/v) acetonitrile
• 10 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate
• 55 mM iodoacetamide in 25 mM ammonium bicarbonate
• 25 mM ammonium bicarbonate, pH 8
• 0.05 to 0.1 mg/ml trypsin (sequence grade; Promega, Pierce) in 25 mM ammonium bicarbonate, pH 8
• 5% (v/v) trifluoroacetic acid (TFA)/50% (v/v) acetonitrile
• 0.65 ml tubes (e.g., PGC Scientific), silanized
• Vacuum centrifuge
• 56°C water bath
• Sonication bath

NOTE: To avoid or reduce contamination with human keratins, one should wear gloves and preferably work in a laminar flow hood.

Excise and wash gel fragments

1. Excise protein bands/spots of interest from a stained polyacrylamide gel. Cut each gel piece into small particles (~1 mm²) using a scalpel, and place into a 0.65 ml siliconized tube. Also cut out and dice a gel piece from a protein free region of the gel, for a parallel control digestion to identify trypsin autoproteolysis products.

   The amount of protein loaded on the gel must be determined empirically as it depends on properties of the specific protein (for example, degree of hydrophobicity and size). In general, picomole levels are preferred to lower levels, although some reports claim that femtomole sensitivities are attainable with in-gel digestion and mass spectrometry.

   The small gel particle size facilitates the removal of SDS (and Coomassie) during the washes, and improves enzyme access to the gel. For Coomassie stained proteins, one gel particle per tube is probably suffictent protein. With faint silver stained spots, particles can be pooled, but no more than three should be pooled as thts will lead to excessive levels of contaminants. For a silver staining protocol compatible with mass spectrometry, see Shevchenko et al. (1996).
    

2. Add ~100 µl of 25 mM ammonium bicarbonate/50% acetonitrile (or enough to immerse the gel particles) and vortex for 10 min. Use gel loading pipet tips to remove the solution (pale blue in the case of Coomassie staining) and discard. Repeat this wash/dehydration step up to ~3 times.

   At this point, the gel slices shrink and become white. This visual criterion should be used to determine whether or not additional washes should be performed.
    

3. 3. Dry gel particles for ~30 min in a vacuum centrifuge.

   Perform reduction and alkylation (optional)
    

4. Add enough 10 mM DTT solution to cover the gel pieces, and reduce for 1 hr at 56ºC.

   Steps 4 to 7 may be included when a maximum of protein coverage is required or when digesting a band from a one dimensional gel. Proteins separated by two dimensional gel electrophoresis are already reduced and alkylated, so these steps can be omitted
    

5. Cool to room temperature and replace the DTT solution with roughly the same volume of 55 mM iodoacetamide solution. Incubate for 45 min at room temperature in the dark with occasional vortexing.
    
6. Wash gel pieces (rehydrate) with ~100 µl of 25 mM ammonium bicarbonate pH 8, for 10 min while vortexing, and dehydrate with ~100 µl of 25 mM ammonium bicarbonate/50% acetonitrile. Repeat rehydration and dehydration.
    
7. Remove the liquid phase and dry the gel pieces in a vacuum centrifuge.

   Digest protein sample
    

8. Rehydrate gel particles in 1 vol of 0.05 to 0.1 mg/ml trypsin solution by vortexing for 5 min. Do not add more solution than can be absorbed by the gel particles.

   The volume needed can be estimated by calculating the total gel volume excised (e.g., 2 mm x 8 mm x 1 mm = 16 mm3 = 16 µl).

   The enzyme substrate ratio employed for in-gel digestions (>1:10) is greater than for in-solution digestions due to hindered enzyme access to the protein substrate in the gel. Moreover, the relatively low salt concentration (25 mM) is needed to reduce the possibility of subsequent salt interference with ionization in the mass spectrometer.
    

9. If necessary, overlay the rehydrated gel particles with a minimum amount of 25 mM ammonium bicarbonate to keep them immersed throughout digestion.
    
10. Incubate 12 to 16 hr at 37ºC.

    Recover peptides
     

11. Add 2 vol water, vortex for 5 min, and then sonicate for 5 min. Use a gel-loading tip to remove the peptide solution and transfer it to a silanized tube.

    Note added by Bruce Stanley: If yields are low, can extract instead with 2 vol saturated (~20 mg/ml) a-hydroxycinnamic acid. Shake gel pieces in this for 1-2 h, then remove liquid and let it air dry in a siliconized tube to ~5-10 µl, then spot onto MALDI plate.
     

12. Perform two additional extractions using 2 vol of 5% TFA/50% acetonitrile.

    The silanized microcentrifuge tubes and the high TFA concentration are used to minimize adsorptive sample loss. Formic acid (5%) may be used as an alternative to TFA.
     

13. Concentrate recovered peptides by reducing the final volume of the extracts to ~10 µl in a vacuum centrifuge. Bring to 25 µl with 5% TFA/50% acetonitrile.

    Store the recovered peptides at -20ºC until MALDI-MS is performed.

    For MALDI-MS analysis, mix 0.5 µl of the unseparated digest on the sample target with 0.5 µl of 20 mg/ml a-hydroxycinnamic acid in 0.1%TFA/ 50% acetonitrile and let air dry. Insert sample target into the mass spectrometer and mass analyze as recommended by the manufacturer.
     

14. Optional: Decrease the amount of volatile salts by adding a few cycles of water (100 to 200 µl) and subsequently reducing the volume in a vacuum centrifuge.

Salt reduction may improve mass signals, particularly at sub-picomole levels.

 

Page maintained by Bruce A. Stanley
Last modified September 03, 2009 02:20 PM          This page has been viewed times since July 2007