Cylindrical gel samples of 1-mmdiameter were excised and destained by incubation with 400 uL
of 25% 2-propanol for 30 min. The destained gel samples were
dried in a Speedvac for 10 min, followed by addition of 5 uL of
digestion buffer (5 mM DTT, 5 mM n-octyl glucopyranoside (n-
OGP), 20 mM Tris, pH 7.8) containing 12 ng/ÌL modified porcine
trypsin (sequencing grade, Promega). After overnight incubation
at 37 °C, 5 ÌL of 0.4% TFA, 5 mM n-OGP was added and the
incubation was extended for 1 h at room temperature. To obtain
sufficient volume for comparison of sample preparation methods,
an additional 5 ÌL of 0.2% TFA, 5 mM n-OGP was added. Samples
were stored at -20 °C prior to MALDI sample preparation.