Premium
Isolation technique and proteomic analysis of the erythrocyte ghosts of red‐eared turtle ( T rachemys scripta )
Author(s) -
Gao Jun,
Li Jianglin,
Feng Can,
Hu Zhaotun,
Liu Wenfeng,
Liang Songping,
Yin Dazhong
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200243
Subject(s) - centrifugation , turtle (robot) , lysis , differential centrifugation , nucleated red blood cell , chromatography , chemistry , digestion (alchemy) , microbiology and biotechnology , biology , biochemistry , genetics , pregnancy , fetus , fishery
To proceed proteomic analysis of erythrocyte of the red‐eared turtle T rachemys 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 T rachemys 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.