[
East Asia Worm Meeting,
2010]
Caenorhabditis briggsae is closely related to C. elegans, which is a model multicellular organism. These 2 species diverged from a common ancestor about 100 million years ago and share several characteristics such as overall morphology, hybridization system, and genes; they differ in terms of optimal growing temperature and intergenic regions. A comparative analysis of these 2 species is expected to generate new evolutionary knowledge. In recent years, in order to understand life phenomena, many proteomic analyses have been performed to exhaustively investigate proteins, which are the primary driving workforces in life phenomena, because the genome cannot be completely understood by using current technology. C. briggsae has rarely been studied in proteomics, although C. elegans has been well studied.In this study, we identified and quantitatively analyzed the expression profiles of proteins expressed during the different developmental stages in C. briggsae-embryo stage, larval 1 stage (L1), and adult stage. Age-synchronized embryos, L1 larvae, and adult individuals of C. briggsae were collected 3 times by using the alkali-bleach method. The collected samples were analyzed using two-dimensional difference gel electrophoresis (2D-DIGE). The spots (proteins) were detected and matched among gels by using image analysis software. We focused on highly reproducible spots, i.e., spots that were detected on at least 2 of all the 3 gels for a particular developmental stage. We obtained 569, 811, and 802 highly reproducible spots in the embryo, L1, and adult stages, respectively; of these spots, 32, 119, and 130 spots were specific to each stage. Ninety proteins expressed at high levels were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The identified proteins were classified on the basis of the Gene Ontology database. Many proteins were found to have ""metabolic process"" and ""cellular process"" functions. To perform a more exhaustive analysis with a higher throughput, we are currently performing a shotgun analysis for the 3 stages of C. briggsae by using LC-MS/MS.
[
International Worm Meeting,
2009]
Protein expression profiles of the developmental stages of Caenorhabditis elegans were determined using matrix-associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-mass spectrometry (LC-MS). The protein expression profiles of the following 6 developmental stages were analyzed: the embryo, 4 larvae, and adult stages. However, these profiles were found to be discrete and not successive. Thus, the elucidation of life systems using systems biology requires quantitative and successive data. In this study, we focused on the protein expression profiles during early embryogenesis in C. elegans because it is known that the cell fate of this organism is determined by the maternal genes expressed up to the 12-cell stage. Worms were synchronized thrice using the alkali-bleach method, and early embryos were collected. The obtained embryos were allowed to develop for 40 minutes, and samples were collected every 10 minutes. The successive samples thus obtained were analyzed using 2-D difference gel electrophoresis (2D-DIGE). As a result, successive data for 521 spots (proteins) were obtained. Among the spots, we assumed that those which showed decreased expression levels were related to cell fate determination. The successive protein expression profiles of the 521 spots were grouped by cluster analysis. Furthermore, several spots were identified using MALDI-TOF MS, and the remaining spots are been identifying using MALDI-TOF MS and LC-MS.
[
International Worm Meeting,
2009]
Caenorhabditis elegans and Caenorhabditis briggsae diverged from a common ancestor roughly 100 million years ago. These 2 Caenorhabditis species differ in some aspects like thermal sensitivity, gene (intergenic region), vulval-uterine, excretory duct and male tail, and at the same time, also share characteristics such as genes and proteins, hybridization system, and morphological body structure. Therefore, we investigated whether or not the expression patterns of gene products were conserved in these species. Comparative proteome analysis between C. elegans and C. briggsae was conducted using two-dimensional difference gel electrophoresis (2D-DIGE). L1 stage larvae of C. elegans and C. briggsae were synthesized using the alkali-bleach method and were collected thrice. Two-dimensional electrophoresis was performed using the Ettan DIGE system and the spots (proteins) were detected using an image analysis software. The spots that showed different expression levels between the 2 species were analyzed using Student''s t-test (p=0.05). In this study, 2D electrophoresis of the 3 obtained samples, showed that 599 spots differed between the 2 species. A total of 301 and 298 spots showed high expression levels in C. elegans and C. briggsae, respectively. In addition, 209 spots-119 spots in C. elegans and 90 spots in C. briggsae-showed a significant difference in Student''s t-test (p=0.05). Although ~95% of the genes were similar between C. elegans and C. briggsae, the protein expression patterns were very different because of the following reasons: (1) difference in amino acid sequences (composition), (2) post-translational modification, and (3) difference in the protein expression pattern. Consequently, to further elucidate these reasons, the proteins were subjected to matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography mass spectrometry (LC-MS).