Webdesign R. Wildgruber

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In-Gel-Digest (prior to MALDI-ToF-MS(MS)

In gel digest process

• 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

Sample Clean up for MALDI-TOF-MS/PMF

• 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

MALDI-Target Sample Deposition

• Recover the peptides directly onto the target plate precoated with Matrix (HCCA recommended)
• Let the sample(s) dry and perform MALDI-TOF-MS

Comments & Hints

• 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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