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

Electrophoresis. 2013 Jan;34(2):215-23. doi: 10.1002/elps.201200243.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Proteins / analysis
  • Blood Proteins / chemistry
  • Cell Separation / methods*
  • Chromatography, Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Erythrocyte Membrane / chemistry*
  • Proteome / analysis
  • Proteome / chemistry*
  • Proteomics / methods*
  • Tandem Mass Spectrometry
  • Turtles / blood*

Substances

  • Blood Proteins
  • Proteome