Patients provided informed consent. Study protocols were approved by the local ethics committee (reg. Number 3882–10) at Ruhr-University Bochum, Bochum, Germany. For each patient 250,000 μm2 of HC, DC, AF or RV tissue was collected by LMD (LMD 6500, Leica Microsystems, Wetzlar, Germany). Sample lysis and digestion were carried out as previously described [25 (link)]. Briefly, samples were lysed with formic acid (98–100%) for 30 min at room temperature (RT), followed by a sonication step for 5 min (RK31, BANDELIN electronic, Berlin, Germany). Samples were kept frozen at − 80 °C until digestion.
Prior to digestion the formic acid was removed and the collected samples were digested in 50 mM ammonium bicarbonate at pH 7.8. Samples were reduced and alkylated by adding dithiothreitol and iodoacetamide. Trypsin (Serva) was added to a final concentration of 1 μg. Digestion was carried out overnight at 37 °C and stopped by adding TFA to acidify the sample. Samples were purified using OMIX C18 Tips (Varian, Agilent Technologies, Böblingen, Germany) completely dried vacuum and again solved in 63 μl 0.1% TFA, as described in [25 (link)].
Prior to digestion the formic acid was removed and the collected samples were digested in 50 mM ammonium bicarbonate at pH 7.8. Samples were reduced and alkylated by adding dithiothreitol and iodoacetamide. Trypsin (Serva) was added to a final concentration of 1 μg. Digestion was carried out overnight at 37 °C and stopped by adding TFA to acidify the sample. Samples were purified using OMIX C18 Tips (Varian, Agilent Technologies, Böblingen, Germany) completely dried vacuum and again solved in 63 μl 0.1% TFA, as described in [25 (link)].
Free full text: Click here
