Sample preparation and protein precipitation

Mass spectrometry (MS) was performed on whole de-yolked stage 40 hatchlings as well as stage 40 eyes. For the whole organism, stage 40 hatchlings were euthanized with tricaine, deyolked, pooled (n=3 biological replicates with n=6 each), snap-frozen in liquid nitrogen and kept at −80°C until lysis. For the measurement of eyes, both left and right eyes were dissected from 15 fish per biological replicate (n=4 biological replicates, 30 eyes each) and snap frozen in liquid nitrogen. Samples were lysed with 100-150 µl of RIPA Lysis and Extraction Buffer including cOmplete EDTA–free Protease Inhibitor Cocktail with the help of Qiagen TissueRaptor II. Protein lysis was incubated on ice with 50 U of benzonase nuclease (Millipore, ,E1014-5KU) for 20 min and at 37°C for 5 min. Samples were then centrifuged at 12,000 g for 10 min at 4°C. Supernatant was taken into fresh tubes and protein concentration was assessed using the Pierce BCA Protein Assay Kit. For protein precipitation, 100 µg and 30 µg of total protein for whole hatchling and eyes, respectively, were used. Samples were precipitated with a methanol/chloroform protocol according to Wessel and Flügge (1984).