ITRAQ-BASED QUANTITATIVE PROTEOMIC ANALYSIS OF HEMOCYTES OF CRASSOSOTREA GIGAS IN RESPONSE TO SYNTHETIC DOUBLE-STRANDED RNA
Keisuke G Takahashi1, 3, Naoki Itoh2 and Katsuyoshi Mori3
1Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University, Sendai 980-0845, Japan
2 Laboratory of Fish Diseases, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
3 Oyster Research Institute, Sendai 981-3217, Japan
e-mail: firstname.lastname@example.org (Keisuke G Takahashi)
Keywords: Pacific oyster, Hemocytes, proteomics, viral RNA, host defense, iTRAQ
The Pacific oyster, Crassostrea gigas, is one of the most important species in worldwide aquaculture production. In this study, we aimed to examine immune response of C. gigas hemocytes against synthetic random double-stranded RNAs (partial sequences of GFP and polyinosinic-polycytidylic acid (poly (I:C) as pathogen-associated molecular patterns (PAMPs). We quantitatively compared the hemocyte proteome profiles using isobaric tags for relative and absolute quantization (iTRAQ)-coupled 2D LC-MS/MS.
The differential proteomes of C. gigas hemocytes were analyzed samples prior to injection with GFP ds RNA or poly (I:C), and at 24 hours post-injection hemocytes. A total of 4798 proteins were identified by LC-MS/MS and sequencing analysis. Using 2.5-fold change in expression as a significant benchmark, 164 (GFP ds RNA) and 148 (poly (I:C) differentially expressed proteins were identified. In the GFP ds RNA-injected group, 82 proteins were up-regulated and 82 proteins were down-regulated. Seventeen differential proteins were involved in immune responses and environmental stress. Up-regulated proteins included three isoforms of C1q-like proteins 4, C1q-TNF-related protein, mollscidin-1 as an antimicrobial peptide and heavy metal-binding protein. Down-regulated proteins included interferon-induced protein L44-like protein, galectin-4, hemagglutinin and HSP 70 protein 12A. Similar results were obtained from the C. gigas hemocytes in the poly (I:C)-injected group.
We also analyzed proteome profiles of the samples at 2 days post injection. We consider that this is the first report of iTRAQ-based proteomic response of C. gigas hemocytes against ds RNA injection.