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Proteins in gel pieces were reduced and alkylated using a final concentration of 5 mM dithithreitol and 15 mM iodoacetamide before digestion with Trypsin (Promega) at 25 ng μl −1 in 50 mM ammonium bicarbonate, first for 1 h on ice and then for 16 h at 37 °C using shaking incubator