Meeting Abstract

P3.12  Saturday, Jan. 5  Validation of Daphnia pulex Genome Annotation by Expression Proteomics CHAN, S.Y.*; KULTZ, D; Univ. of California, Davis sychan@ucdavis.edu

We performed an experimental analysis of the Daphnia pulex proteome to validate D. pulex genome annotations. D. pulex were harvested in log growth phase, homogenized and proteins extracted in an aqueous buffer. Electrophoresis of proteins was accomplished on SDS-PAGE gels, followed by the staining and cutting of gel bands according to molecular weight and in-gel digestion with trypsin. Peptides present in each molecular weight fraction were purified by reversed phase liquid chromatography using C18 and then sequenced by MALDI-ToF/ToF mass spectrometry. From these sequences, over 900 unique proteins were identified by LC-MALDI analysis and ProBLAST matching against the D. pulex annotated genome containing 25477 protein entries (minimum number of sequenced peptides =3 and minimum overall ProBLAST score =120). Gene annotations were considered valid if their predicted molecular mass matched the observed molecular mass of expressed proteins. In cases where these masses do not match, partial proteolysis of proteins in samples or incomplete sequence annotation are possible causes. In addition to validating genome annotations by expression proteomics, our proteomics data enabled identification of major biological processes and cellular functions in log phase D. pulex using PANTHER analysis, and demonstrated the feasibility of high throughput proteomics of D. pulex.