Retinal Protein Extraction and Trypsin Digestion

Each isolated retina was prepared first by incubating in 200 µL 8 M aqueous urea solution for 30 min followed by centrifugation for 5 min at 1400× g [20 (link),23 (link)]. The supernatant was collected, and protein content was measured using a microplate reader (BioTek Synergy H1 with Take3 plate; Agilent, Santa Clara, CA, USA). An aliquot containing 100 µg protein was taken and the volume was adjusted with 25 mM ammonium bicarbonate to 100 µL. The sample was then reduced with dithiothreitol and carbamidomethylated with iodoacetic amide [20 (link),23 (link)]. After 9-fold dilution of the sample with aqueous 25 mM ammonium bicarbonate, trypsin digestion was performed at 37 °C overnight, then the reaction was quenched by acidification with formic acid (1 µL). The solution was desalted by solid-phase extraction (SPE) using 1 mL Sep-Pak™ C18 SPE cartridges (Waters, Milford, MA, USA), and the extract was dried under vacuum (Vacufuge™, Eppendorf AG, Hamburg, Germany) into a 1.5 mL centrifuge tube.