Protocols

MALDI mass spectrometry analysis

Preparation of the samples for MALDI mass spectrometry analysis

• add 100 µl Buffer 2 (2 ml, 70 % 50 mM AB (ammoniumbicarbonate), 30 % ACN (acetonitrile) to the Coomassie colored SDS gelpiece, incubate for 30 min, RT
• remove Buffer 2, add 100 µl ACN and incubate for 10 min, RT
• remove ACN, add 100 µl DTT (10 mM dithiothreitol and 50 mM ammoniumhydrogencarbonate),
  incubate for 45 min, 56 °C
• remove DTT, add 100 µl IAA (55 mM iodacetamide and 50 mM AB), incubate for 30 min at RT with exclusion of light
• remove IAA, add 100 µl Buffer 1 (50 mM AB), incubate for 15 min, RT
• remove Buffer 1, add 100 µl Buffer 2, incubate for 10 min, RT
• remove Buffer 2, add 100 µl ACN, incubate for 10 min, RT
• remove ACN, add 20 µl Trypsin (β= 8.3 ng/µl)
• after 6 h add 2 µl 10 % TFA (trifluoracetate) to stop digestion

Measurement of the samples

• add 1 µl matrix to goldplate, after that add 1 µl probe and mix them
• after drying insert goldplate to MALDI massspec