Protein extraction from tissue biopsies and microdialysate
Tissue samples were solubilized in urea and NaCl with ProteaseMAX (Promega) in ammonium bicarbonate (AmBic) and mixed vigorously. Furthermore, samples were quickly frozen on block and subjected to disruption with a Vortex Genie disruptor before additional NaCl, urea and AmBic were mixed in. The supernatant fraction was transferred to new tubes after centrifugation at 10000 x g and at 4°C, AmBic was again added, and the tubes were vortexed vigorously for 2 minutes.For assessment of microdialysates, the proteins were reduced with dithiothreitol in AmBic and then alkylated with iodoacetamide in AmBic in the absence of light. Trypsin (sequencing grade, Promega) was used to prepare proteolytic digestions of microdialysate proteins overnight. After adding concentrated formic acid, the reaction was stopped. Samples were subsequently cleaned and dried using a vacuum concentrator (miVac, Thermo scientific).