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 無 50 mM ammonium bicarbonate/ 30% acetonitrile
  • Incubate at 37蚓 5'
  • Discard Supernatant
  • Optional: Reduce & alkylate with:
  • 5 無 10 mM TCEP (Tris(2-carboxyethyl)phosphine HCl) in 50 mM ammonium bicarbonate/ 30% acetonitrile
  • Mix and incubate 30 min at ambient temperature
  • 5 無 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 50痞 80% acetonitrile
  • Incubate at ambient temperature 10'
  • Discard Supernatant
  • Carefully dry off residual liquid at 37蚓
  • Initiate digest by addition of 2無 of trypsin (12.5 ng/無) to the gelplug
  • Wait 5 min for gel to swell
  • Add 4無 5mM Tris-HCl, pH 8.0
  • 1 hour at 37蚓
  • Terminate digest by addition of 12痞 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 10痞 0.1% TFA / 80% acetonitrile
  • 2 x 10痞 0.1% TFA
  • Bind solubilized peptides to SPE-resin
  • Pipette up and down 10 times to ensure adsorption
  • Wash bound peptides with 2 x 10痞 0.1% TFA
  • Recover peptides by aspirating 2痞 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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