Protocols

MALDI mass spectrometry analysis

Preparation of the samples for MALDI mass spectrometry

1. 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
2. remove Buffer 2, add 100 µl ACN and incubate for 10 min, RT
3. remove ACN, add 100 µl DTT (10 mM dithiothreitol and 50 mM ammoniumhydrogencarbonate), incubate for 45 min, 56 °C
4. remove DTT, add 100 µl IAA (55 mM iodacetamide and 50 mM AB), incubate for 30 min at RT with exclusion of light
5. remove IAA, add 100 µl Buffer 1 (50 mM AB), incubate for 15 min, RT
6. remove Buffer 1, add 100 µl Buffer 2, incubate for 10 min, RT
7. remove Buffer 2, add 100 µl ACN, incubate for 10 min, RT
8. remove ACN, add 20 µl Trypsin (β= 8,3 ng/µl)
9. after 6 h add 2 µl 10 % TFA (trifluoracetate) to stop digestion

Measurement of the samples

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