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.
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