FIGURE 1.

Proteome analysis of zebrafish myelin. (A) One-dimensional separation of myelin biochemically purified from brains and optic nerves of adult zebrafish. Myelin was separated by 1D SDS-PAGE on Tris-glycine gradient gels, and proteins were visualized by silver staining (0.5 μg load) or colloidal Coomassie (CBB250; 5 μg load). Bands are annotated that mainly comprise known myelin proteins according to mass spectrometric identification. Arrowheads indicate bands in which no known myelin proteins were identified. (B) Myelin separated on Tris-glycine gradient gels (5 μg load) before (pre-wash) or after (post-wash) an additional step of high-pH and high-salt conditions. The indicated grid divides each CBB250-stained lane into equally sized slices, which were excised for automated tryptic digest and LC-MS analysis, thereby, respectively, identifying 890 (pre-wash) and 1274 (post-wash) proteins (Supplementary Table 1). (C) Number and relative abundance of proteins identified and quantified in purified myelin by in-solution digestion and two data independent acquisition MS modes, MS^E and UDMS^E. Note that MS^E (orange) identifies fewer proteins but provides a higher dynamic range for quantification of proteins in purified myelin compared to UDMS^E. MS^E thus facilitates quantification of highly abundant myelin proteins. ppm, parts per million. (D) Venn diagram comparing the number of proteins identified in myelin by MS^E, UDMS^E, and gel-based approaches.