The Rockefeller University
1230 York Avenue,
New York, NY 10065
for the Mass Spectrometric
Analysis of Biological
MALDI sample preparation: in-gel digestion and crystallization
Protocol was developed by J DeGrasse, M. Kalkum, A. Krutchinsky, J.C. Padovan and W. Zhang
- Electrophoresis solutions, such as running buffers and sample loading buffers, may be prepared with milliQ-grade water. All other solutions should be made with HPLC-grade solvents.
- 100mM ammonium bicarbonate. 3.95g ammonium bicarbonate into 500ml water. Filter with a 0.22µm size filter. Store at 4°C.
- 200mM dithiothreitol. 30.9mg dithiothreitol into 1ml of 1mM aqueous HCl solution. Aliquot. Store at -20°C.
- 1M iodoacetamide. 46.3mg into 250µl water. Aliquot. Store at -20°C.
- Destaining solution. 50mM ammonium bicarbonate in 50% methanol / 50% water. Store at 4°C.
- Trypsin. Add 25µl of 1mM HCl to the lyophilized trypsin vial (Roche #11418025001). Separate into 1, 2 or 5-µl aliquots. Store at -20°C.
- POROS bead slurry (50µg/µl in 20% ethanol) (POROS 20 R2, Applied Biosystems). Dry stock POROS beads are washed in equal volumes of (1) methanol, then (2) 80% acetonitrile, then (3) 20% ethanol and then (4) resuspended in 20% ethanol at 50µg/µl and stored at 4°C.
- 10% (w/v) trifluoroacetic acid (from Pierce #28902).
- Elution solutions
- DHB elution solution: 20% acetonitrile, 50% methanol, and 0.1% trifluoroacetic acid.
- 4-HCCA elution solution: 70% acetonitrile, 0.1% trifluoroacetic acid.
- Calibrants. Bradykinin (904Da), Neurotensin (1672Da), oxidized Insulin ▀-chain (3496Da). To a 10µl 50µM stock solution (stored at -80°C), add 90µl 50% acetonitrile, 5% ethanol, 45% water. Store the 5µM stock at -20°C.
- Dilute the protein sample in gel-loading buffer. (We suggest bis-Tris gel systems, Invitrogen)
- 2.5µl NuPAGE« LDS (lithium dodecyl sulfate) Sample Buffer (#NP0007).
- 1µl 200mM dithiothreitol (DTT).
- Bring to 10µl with sample and/or water.
- Heat at 70║C for 10 minutes (with agitation, if possible), allow to cool to room temperature.
- To alkylate the reduced cysteines, add 1µl of 1M iodoacetamide per 10 µL of gel loading buffer. React in the dark for 30 minutes at room temperature.
- After running the gel, stain with GelCode Blue (Pierce, catalog number 24592). After recording an image of the gel, destain thoroughly to remove most of the background.
- Excise the bands of interest using tweezers and a fine scalpel (fine-point diamond tweezers and 15- or 30-degree feather microscalpel, Electron Microscopy Sciences).
- Alternatively, one could remove the lane from the gel and cut 2-4mm slices down the length of the lane. This allows for the detection of low-abundance proteins that may not be detected by staining.
- Dice each slice into 1mm3 using either the scalpel or the tweezers. Transfer the cubes to a microcentrifuge vial filled with 500µl of destaining solution.
- Washing (and destaining) gel slices
- Shake the vials in a microvial mixer (Microtube Mixer, MT-360, TOMY Tech USA) at 4°C.
- Aspirate the destaining solution and replace with fresh solution every 30 minutes and wash for 4 hours or until all traces of Coomassie have been removed. Residual amounts of Coomassie and detergents (e.g., SDS, MOPS, etc.) will partially inhibit trypsin digestion. Also, the Coomassie will co-extract with the peptides.
- After the final aspiration, add 100µl acetonitrile to dehydrate the gel pieces. Aspirate the liquid. Air-dry for 5 minutes.
- One can leave the dried gel pieces overnight at 4°C.
- Prepare trypsin aliquot
- Add 19µl 50mM ABC to a 1µl trypsin aliquot (50ng/µl final concentration).
- Add 75-150ng of trypsin to the dried gel pieces and allow pieces to swell (~5min).
- Top off with 40µl of 50mM ABC in 10% acetonitrile. Be sure gel pieces are covered.
- Digest at 37°C for 4-6 hours. Four hours is normally sufficient.
- Prepare POROS beads
- 1 volume stock POROS bead slurry.
- 9 volumes 5% formic acid, 0.2% trifluoroacetic acid(aq).
- Add 40µl of slurry to each digestion vial (or the equivalent of the digestion volume).
- Incubate on the microvial mixer at the highest setting. 4 hours at 4°C.
- ZipTip Conditioning and Sample Loading
- Prepare ZipTips
- Wash ZipTips with:
- 2 x 10µl 0.1% trifluoroacetic acid.
- 4 x 10µl DHB or 4-HCCA elution solution.
- 4 x 10µl 0.1% trifluoroacetic acid.
- leave 10µl 0.1% trifluoroacetic acid to keep tips wet, and remove prior to use.
- Transfer the 80-ml digest and bead mixture to the cleaned ZipTips and spin the liquid through for 1-2min at 1000rpm. Discard the flowthrough.
- Wash gel pieces in microcentrifuge vial with 20µl 0.1% trifluoroacetic acid, transfer wash onto ZipTip and expel the wash solution as above.
- Thoroughly wash beads 2x with 20µl 0.1% trifluoroacetic acid. Expel wash solution as above.
- One can leave the ZipTips overnight at 4°C if the beads are kept wet with 0.1% trifluoroacetic acid.
- Elution Option A: 2,5 dihydroxybenzoic acid (DHB)
- Prepare matrix
- To solid DHB (fill a 1.5ml microcentrifuge vial to the 100µl mark), add 50µl of DHB elution solution. Vortex for 5min or until the solution is saturated. Spin at 14k rpm for 5 minutes.
- In a fresh vial, dilute the saturated solution 1 in 3 (33% saturated DHB) with DHB elution solution.
- Place 2.5µl of the 33% saturated DHB onto the top of the ZipTip behind the POROS beads. Slowly elute onto the sample plate.
- Elution option B: a-Cyano-4-hydroxycinnamic acid (4-HCCA)
- Prepare Matrix
- To a solid 4-HCCA aliquot, add 70µl acetonitrile and vortex. Use a pipet tip to dislodge the solid from the walls of the vial. Vortex for 2 minutes.
- Add 30µl of water. Vortex for 2 minutes.
- Add trifluoroacetic acid to 0.1% final concentration.
- Spin at 14k rpm for 5 minutes.
- In a fresh vial, dilute the saturated solution 1 to 1 (50% saturated 4-HCCA) with 4-HCCA elution solution.
- Place 2.5-3.0µl of the 50% saturated 4-HCCA onto the top of the ZipTip behind the POROS beads. Slowly elute onto the sample plate.
- If desired, the crystallized spots may be washed with cold 0.1% TFA, which is immediately aspirated.
- Dilute the 5pmol/µl stock 1 in 10 in 50% acetonitrile, 5% ethanol, 45% water to produce a 500 fmol/µL solution.
- Dilute the 500fmol/µl solution 1 in 5 with matrix (33% DHB or 50% 4-HCCA) and spot 1-2µl spots in close proximity to your sample. One calibrant spot for every 10 sample spots is sufficient.