1.proteome Comparative Analysis Of Gynogenetic Haploid Embryos With Normal Eyes And Cupless Eyes Of Goldfish 2.application Of Mass Spectrometry Driven Blast In Protein Identification Of Goldfish Embryos

PartⅠ: Proteome comparative analysis of gynogenetic haploid embryos with normal eyes and cupless eyes of goldfishOur previous work indicated that obstruction of some protein expression cause the abnormal development of haploid embryos, such as the protein Vsx1(Huang LY et al). Further studies on Vsx1 confirmed the diploid development regulatory mechanism of gene expression during embryonic morphogenesis of goldfish (Luo et al) .The two genes of Vsx1 are expressed in gynogenetic haploid embryos with normal eyes of goldfish, while not with cupless eyes at all.To better understand the molecular events underlying the processes occurring at the embryos developing of gynogenetic haploid and elucidate the underlying molecular mechanism of abnormal development in haploid embryos, we developed a proteome comparative analysis of the gynogenetic haploid embryos with normal eyes and cupless eyes of goldfish in the same stage to disclose more differentially expressed proteins as Vsx1. The results indicated protein profiles of embryos with normal eyes and with cupless eyes exhibited some differences. Fifty-two protein spots were directly identified by mass spectrometry and mascot search. Seven protein spots were identified by sequence similarity search using mass spectrum driven BLAST (MS BLAST). Western blot analysis was further carried out to verify the different expression of four important proteins, which offered different significantly gene expression during embryos development between haploid embryos with normal eyes and with cupless eyes.PartⅡ: The application of Mass Spectrometry driven BLAST in protein identification of goldfish embryosProtein identification is a significant bottleneck in proteomic researches of some species with unknown genomes or uncompleted protein database at present. Mass Spectrometry driven BLAST (MS BLAST) is a sequence similarity searching tool, which has been developed in recent years and successfully applied to the identification of protein from species with unsequenced genomes. SPITC derivation is an improved de novo sequencing method by Mass Spectrometry. Combining these two methods, nineteen proteins of goldfish embryos not identified by Mascot searching were successfully identified by MS BLAST. Among the nineteen proteins, twelve were identified by MS BLAST searching, while the other seven by MS BLAST searching based on de novo sequencing of SPITC derivated peptides. This strategy has been proved feasible and reliable for protein identification of goldfish embryos, and provides a new pathway for protein cross-species identification of species with unsequenced genomes.