Mtp 384 ground steel maldi target plate

Sample preparation for MALDI-TOF MS analysis was performed as follows: the culture medium was discarded from the flasks; the cells were washed with 10 ml PBS and were subsequently incubated with 3 ml 0.025% trypsin/EDTA for 3–5 min. The reaction was terminated by the addition of 10 ml cell culture medium. The cells were stained with trypan blue and the cell number was established using the Neubauer cell counting chamber. The suspension was subsequently centrifuged at 1,700 × g for 5 min. The supernatant was discarded and the cell pellet was mixed with 1 ml of ethanol. Following centrifugation at 1,700 × g for 5 min, the ethanol was removed and the pellet was reconstituted in 70% formic acid at a ratio of 20 µl/1×10⁶ cells. The mixture was left at room temperature for 2 min, and then an equal volume of acetonitrile was added. The samples were then centrifuged at 10,000 × g for 5 min. An aliquot (1 µl) of the supernatant was spotted in duplicate onto the MTP 384 ground steel MALDI target plate (Bruker Daltonics). The sample was allowed to dry at ambient temperature and then overlaid with 1 µl of HCCA (5 mg/ml in a mixture of acetonitrile, water and trifluoroacetic acid 50:47.5:2.5% v/v).