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Isolation technique and proteomic analysis of the erythrocyte ghosts of red‐eared turtle (Trachemys scripta)

To proceed proteomic analysis of erythrocyte of the red‐eared turtle Trachemys scripta, a method for obtaining turtle erythrocyte ghosts (TEG) was first developed. After hypotonic lysis using a special buffer, forcing the erythrocyte through the syringe with an “N”‐shaped needle, applying low speed homogenizing and differential centrifugation, highly purified TEG fractions were obtained. The isolated TEG proteins were treated with in‐gel digestion separated by SDS‐PAGE or in‐solution digestion followed by HPLC predissociation, and then identified by nano‐ESI‐LC MS/MS techniques. A total of 169 TEG proteins was identified, validated, and categorized. Among these proteins, tubulins, and cell‐surface‐located F‐type ATP synthase revealed important information into the super tolerance of Trachemys scripta in anoxia and low temperature exposure. Altogether, this study not only provided a method to isolate high quality TEG and a dataset of comprehensive characterization of TEG proteins, but also provides a tool for proteomic research of all nucleated red blood cells, and thus opened a new research field for exploring the mechanisms of super tolerance of turtles in harsh environment.