Comparative Proteomic Analysis of Rana chensinensis Oviduct
As one of most important traditional Chinese medicine resources, the oviduct of female Rana chensinensis (Chinese brown frog) was widely used in the treatment of asthenia after sickness or delivery, deficiency in vigor, palpitation, and insomnia. Unlike other vertebrates, the oviduct of Rana chensinensis oviduct significantly expands during prehibernation, in contrast to the breeding period. To explain this phenomenon at the molecular level, the protein expression profiles of Rana chensinensis
... Rana chensinensis oviduct during the breeding period and prehibernation were observed using isobaric tags for relative and absolute quantitation (iTRAQ) technique. Then, all identified proteins were used to obtain gene ontology (GO) annotation. Ultimately, KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was performed to predict the pathway on differentially expressed proteins (DEPs). A total of 4479 proteins were identified, and 312 of them presented different expression profiling between prehibernation and breeding period. Compared with prehibernation group, 86 proteins were upregulated, and 226 proteins were downregulated in breeding period. After KEGG enrichment analysis, 163 DEPs were involved in 6 pathways, which were lysosome, RNA transport, glycosaminoglycan degradation, extracellular matrix (ECM)-receptor interaction, metabolic pathways and focal adhesion. This is the first report on the protein profiling of Rana chensinensis oviduct during the breeding period and prehibernation. Results show that this distinctive physiological phenomenon of Rana chensinensis oviduct was mainly involved in ECM-receptor interaction, metabolic pathways, and focal adhesion. The hibernation for Rana chensinensis ranges from October to February next year, followed by the breeding period from February to June. After the breeding period, Rana chensinensis goes into the prehibernation period until October  . Distinguishing from the oviduct expansion during the breeding period in other species, a unique physiological phenomenon of Rana chensinensis is that its oviduct starts to expand after breeding, reaching a peak by October during the prehibernation  . For this reason, Rana chensinensis oviduct, as traditional Chinese medicine, was collected from frog in the autumn before hibernation. To ascertain the signaling pathways involved in the timing of oviduct expansion, we should clarify the changes of macromolecular components between prehibernation and breeding period. Proteomics is an efficient methodology to analyze protein expression, and it will disclose protein expression profiles in different physiological phases  . It is widely used to tackle biological problems, whereby the raw data obtained from proteomics is studied further by bioinformatic methodologies  . Isobaric tags for relative and absolute quantitation (iTRAQ) is an isobaric labeling method applied in quantitative proteomics by tandem mass spectrometry to identify the amount of proteins from different samples in a single experiment  . iTRAQ can separate and identify a variety of proteins, including membrane proteins, proteins of high molecular weight, insoluble proteins, acidic proteins, and alkaline proteins . Results Protein Identified through Itraq Technology 13216 unique peptides and 4479 proteins were identified using the iTRAQ technology (see Tables S1 and S2 in Supplementary Material). Among these identified proteins, 2422 were 0-20 kDa, 1758 were 20-60 kDa, and 299 were over 60 kDa ( Figure 1A) . Accordingly, we found that identified proteins were primarily below 20 kDa. To further prove the credibility of protein identification, peptide sequence coverage and the unique peptide numbers of proteins were also regarded as two important quality evaluation parameters. Figure 1B showed that peptide sequence coverage of these proteins was basically less than 30%. The number of unique peptides for identified proteins were mainly concentrated in 1 and 2, which makes up approximately 71% of the total unique peptides ( Figure 1C ).