The zebrafish lens proteome during development and aging

PURPOSE Changes in lens protein expression during zebrafish development results in a smooth gradient of refractive index necessary for excellent optical function. Age-related changes in crystallin expression have been well documented in mammals but are poorly understood in the zebrafish. METHODS In the zebrafish lens, a systematic analysis of protein content with age was performed using size exclusion chromatography (SEC) combined with linear trap quadrupole Fourier transform tandem mass spectrometry (LTQ-FT LC-MS/MS; rank-order shotgun) proteomics in lenses of larval, juvenile, and adult zebrafish. RESULTS alpha-Crystallins, previously shown to have low abundance in the zebrafish lens, were found to increase dramatically with maturation and aging. SEC determined that beta-crystallin was predominant at 4.5 days. With age, the alpha- and gamma-crystallins increased, and a high molecular weight fraction appeared between six weeks and six months to become the dominant component by 2.5 years. Similarly, shotgun proteomics determined that beta-crystallins were the predominant proteins in the young lens. With age, the proportion of alpha- and gamma-crystallins increased dramatically. After crystallins, calpain 3, membrane, and cytoskeletal proteins were most abundant. Five new beta-crystallins and 13 new gamma-crystallins were identified. CONCLUSIONS As expected, SEC and proteomics demonstrated changing levels of protein expression with age, especially among the crystallins. The results also confirmed the existence of novel crystallins in the zebrafish genome.