Electrophoresis 2013, 34, 215–223 215 Jun Gao * Jianglin Li * Can Feng Zhaotun Hu Wenfeng Liu Songping Liang Dazhong Yin College of Life Sciences, Hunan Normal University, Changsha, Hunan, China Received April 24, 2012 Revised July 29, 2012 Accepted August 9, 2012 Research Article 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 iden- tified, 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. Keywords: Erythrocyte ghost / LC-MS/MS / Proteomics / Red-eared turtle DOI 10.1002/elps.201200243  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Turtles are known as living fossils with unusual physiology, particularly longevity. Although their remarkable character- istics of tolerance to harsh environment (such as low temper- ature, hunger, and hypoxia) have been investigated for about half a century, the exact physiological reason for their tol- erance still remains in puzzle. Uncovering the mechanisms of their super tolerance is of great biological importance, such as, preserving isolated organs for a long time in organ transplantation, avoiding the free-radical damages during the process of reoxygenation and reperfusion, etc. With the advantages of being easy to obtain and lack of in- ternal organelles, human red blood cells (RBC) have been in- vestigated intensively during the past century. The structures and the functions of human erythrocyte proteins are known in great details [1]. Recently, MS-based proteomics has pro- Correspondence: Professor Dazhong Yin, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China E-mail: dazhongyin002@126.com Fax: +86-731-8887-2786 Abbreviations: EM, electron microscopy; HEG, human ery- throcyte ghosts; HLB, hypotonic lysis buffer; RBC, red blood cells; TEG, turtle erythrocyte ghosts vided unprecedented information about mammal RBC [1–4]. RBC ghost proteomics is of particular interest because it has more potential significance on cell protection, material trans- port, signal transduction, and as drug targets, than the whole blood cells [4, 5]. Morphology and biochemistry of turtle erythrocytes have been investigated for about half a century. Turtle erythro- cytes are oval, translucent, approximately 10 times larger than human RBC, and they contain nuclei and almost ab- sent from subcellular organelles [6]. Previous studies have reported some evidences for their important functions when faced with adverse environments [7–9]. Turtle erythrocytes have been used as models for metabolism, evolution, and de- velopment research, etc [10–13]. Nevertheless, we still know very little about the structure and composition of turtle ery- throcytes. In particular, we know very little about erythrocyte ghost proteomics. Previous studies introduced some ghost isolation method of nucleated erythrocytes, such as hypotonic lysis, hyper acoustic break, and differential centrifugation. Although these isolated ghosts were pure enough to study the ultrastructure, cytoskeletal organization, major plasma membrane proteins, and phospholipids [14, 15], they did not ∗ These authors contributed equally to this work. Colour Online: See the article online to view Fig. 2 in colour. C  2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com