Preparation of intact mitochondria using free-flow isoelectric focusing with post-pH gradient sample injection for morphological, functional and proteomics studies

Yu Chen He, Fan Zhi Kong, Liu Yin Fan, Jane Y. Wu, Xiao Ping Liu, Jun Li, Yan Sun, Qiang Zhang, Ying Yang, Xue Jing Wu, Hua Xiao, Cheng Xi Cao*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Mitochondria play essential roles in both energy metabolism and cell signaling, which are critical for cell survival. Although significant efforts have been invested in understanding mitochondrial biology, methods for intact mitochondria preparation are technically challenging and remain to be improved. New methods for heterogeneous mitochondria purification will therefore boost our understanding on their physiological and biophysical properties. Herein, we developed a novel recycling free-flow isoelectric focusing (RFFIEF) with post-pH gradient sample injection (post-PGSI) for preparative separation of mitochondria. Crude mitochondria of rabbit liver obtained from differential centrifugation were purified by the developed method according to their pI values as six fractions. Transmission electron microscope images revealed that intact mitochondria existed in two fractions of pH 6.24 and 6.61, degenerative mitochondria were in two fractions of pH 5.46 and 5.72, and inner membrane vesicles (IMVs) appeared in the fractions of pH 4.70 and 5.04. Membrane potential measurement proved a dramatic difference between intact mitochondria and IMVs, which reflected the bioactivity of obtained populations. Particularly, proteomics analyses revealed that more number of proteins were identified in the intact fractions than that of IMVs or crude mitochondria, which demonstrated that RFFIEF could be powerful tool for the preparation of intact organelle as well as their proteomic and in-depth biological analysis.

Original languageEnglish (US)
Pages (from-to)200-208
Number of pages9
JournalAnalytica Chimica Acta
Volume982
DOIs
StatePublished - Aug 22 2017

Keywords

  • Free-flow electrophoresis
  • Isoelectric focusing
  • Mitochondria
  • Proteomics
  • Purification

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy

Fingerprint Dive into the research topics of 'Preparation of intact mitochondria using free-flow isoelectric focusing with post-pH gradient sample injection for morphological, functional and proteomics studies'. Together they form a unique fingerprint.

Cite this