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In-Gel-Digest (prior to MALDI-ToF-MS(MS)
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In gel digest process
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- Wash Gelplug with 2 x 50 µL 50 mM ammonium bicarbonate/ 30% acetonitrile
- Incubate at 37°C 5'
- Discard Supernatant
- Optional: Reduce & alkylate with:
- 5 µL 10 mM TCEP (Tris(2-carboxyethyl)phosphine HCl) in 50 mM ammonium bicarbonate/ 30% acetonitrile
- Mix and incubate 30 min at ambient temperature
- 5 µL 40 mM iodoacetamide in 50 mM ammonium bicarbonate/ 30% acetonitrile
- Mix and wait 30 min at RT in the dark
- Remove solution and wash with 50 mM ammonium bicarbonate/ 30% acetonitrile
- Dehydrate Gelplug with 50µl 80% acetonitrile
- Incubate at ambient temperature 10'
- Discard Supernatant
- Carefully dry off residual liquid at 37°C
- Initiate digest by addition of 2µL of trypsin (12.5 ng/µL) to the gelplug
- Wait 5 min for gel to swell
- Add 4µL 5mM Tris-HCl, pH 8.0
- 1 hour at 37°C
- Terminate digest by addition of 12µl 1% TFA
- Wait 15' at ambient temperature
- Digest is now ready for HPLC/MS or sample clean up for MALDI-TOF-MS/PMF analysis
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Sample Clean up for MALDI-TOF-MS/PMF
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- Equilibrate SPE-Pipette Tip by aspirating up and down :
- 2 x 10µl 0.1% TFA / 80% acetonitrile
- 2 x 10µl 0.1% TFA
- Bind solubilized peptides to SPE-resin
- Pipette up and down 10 times to ensure adsorption
- Wash bound peptides with 2 x 10µl 0.1% TFA
- Recover peptides by aspirating 2µl 0.1% TFA / 80% acetonitrile
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MALDI-Target Sample Deposition
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- Recover the peptides directly onto the target plate precoated with Matrix (HCCA recommended)
- Let the sample(s) dry and perform MALDI-TOF-MS
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Comments & Hints
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- Keeping the incubation times as short as possible is necessary for maximum performance
- Sensitivity is concentration dependent: (1) Digest in as little volume as possible, (2) recover peptides in minimal volume, (3) avoid repeated transfers of peptides (minimize surface contacts).
- MALDI supression by excessive amount of trypsin will obscure low abundant signals (use as little trypsin as possible)
- Keep the gel-plugs as small as approx. 1mm2
- MALDI Matrix preparation: dissovle 0.18 g/L a-hydroxy-cinnamic acid (HCCA) in 90% acetonitrile/ 0.1% TFA. Use only purest quality HCCA
- The protocol is suited for any gelsystem used to separate proteins, disulphide reduction-alkylation is optional
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