Retinal Protein Solubilization and Digestion

To dissolve proteins of the retinae, each isolated retina was incubated in 200 µL of 8 M aqueous urea solution for 60 min, and subsequently centrifuged for 5 min at 1400 g, as reported before [13 (link),34 (link)]. After collecting the supernatant, protein contents were estimated using a Microplate reader (BioTek Synergy H1 with Take3 plates, Agilent, Palo Alto, CA, USA), and the samples were aliquoted to contain 100 µg protein each. The volume of the aliquoted samples was adjusted to 100 µL with 25 mm NH₄HCO₃ solution followed by reduction with dithiothreitol (1 mM at 65 °C for 30 min) and carbamidomethylation (5 mM iodoacetamide at room temperature and in the dark for 30 min). After 9-fold dilution of the reduced and alkylated sample with aqueous 25 mM ammonium bicarbonate solution, sequencing-grade trypsin (2 µg) was added to digest the proteins overnight at 37 °C, after which the digestion was quenched with 5 µL of formic acid. The samples were desalted by solid-phase extraction using 1-mL Sep-Pak™ C-18 cartridges (Waters USA, Milford, MA), and then the extracts were dried under vacuum (Vacufuge™, Eppendorf AG, Hamburg, Germany) into 1.5-mL centrifuge tubes. The dried residues were dissolved in an aqueous medium containing 5% (v/v) and 0.1% (v/v) formic acid to produce 1 µg/µL protein content.