iTRAQ-based proteomic analysis of adaptive response in the regenerating limb of the Cynops orientalis newt

Abstract

The newt has the powerful capacity to regenerate lost limbs following amputation, and represents an excellent model organism to study regenerative processes. However, the molecular basis of the adaptive response in the regenerating limb of the Chinese fire-bellied newt Cynops orientalis immediately after amputation remains unclear. To better understand the adaptive response immediately after limb amputation at the protein level, we used isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS methods to analyze changes in the proteome of the regenerating newt limb that occurred 2 h and 8 h after amputation. We identified 152 proteins with more than 1.5-fold change in expression compared to control. GO annotation analysis classified these proteins into several categories such as signaling, Ca 2+ binding and translocation, transcription and translation, immune response, cell death, cytoskeleton, metabolism, etc. Further ingenuity pathway analysis (IPA) showed that several signaling pathways were significantly changed at 2 h and 8 h after amputation, including EIF2 signaling, acute phase response signaling, tight junction signaling and calcium signaling, suggesting these pathways may be closely related to the adaptive response immediately after limb amputation. This work provides novel insights into understanding the molecular processes related to newt limb regeneration immediately after amputation, and a basis for further study of regenerative medicine. KEY WORDS: Cynops orientalis, limb regeneration, proteomic, stress response, cell deathAmong vertebrates, urodele salamanders possess remarkable capability to regenerate appendages from any level of amputation through blastema formation. Subsequently, blastema cells that morphologically resemble mesenchymal stem-like cells self-organize into the amputated limb parts , Nye et al., 2003. Adult newt has already been used as an important model for the limb regeneration studies. The process of limb regeneration can be divided into three major phases: wound healing and dedifferentiation; blastema accumulation and blastema growth; differentiation and morphogenesis (Iten and Bryant, 1973). Following amputation, the wound surface is covered rapidly by epithelial cells, which form the wound epidermis at the end of the stump. More importantly, a specialized epithelium provides signals to the underlying cells of Int. J. Dev. Biol. 59: 487-496 (2015) Abbreviations used in this paper: iTRAQ , isobaric tags for relative and absolute quantitation; IPA, ingenuity pathway analysis.

Comparative iTRAQ proteomics revealed proteins associated with lobed fin regeneration in Bichirs

“…In a recent study, through the iTRAQ method, Tang, J., et al found that differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding in the salamanders’ blastema formation [40]. By similar iTRAQ method, Geng et al found that in the early stages of newt’s blastema formation, differentially expressed proteins were concentrated in the categories such as signaling, Ca 2+ binding and translocation, transcription and translation, immune response, cell death, cytoskeleton, metabolism [41]. Correspondingly, in the current study, DEPs are mainly concentrated in ribosome and RNA transport, metabolic, ECM-receptor interaction, Golgi and endoplasmic reticulum, DNA replication, and Regulation of actin cytoskeleton, suggesting that these biological processes are active during the appendage regeneration process.…”

Proteomic analysis of eleven tissues in the Chinese giant salamander (Andrias davidianus)

“…Protein extraction was performed using a procedure described previously 30 . Briefly, the frozen tissue was homogenized, ultrasonically disrupted and centrifuged, and the supernatant was collected.…”

“…Strong cation exchange chromatography and protein identification by mass spectrometry were performed according to the method previously described 30,32 . A total of 33 fractions were collected over the gradient, but some were pooled to give a final total of 15 fractions based on the SCX chromatogram.…”