Expanding the subproteome of the inner mitochondria using protein separation technologies: One-and two-dimensional liquid chromatography and two-dimensional gel electrophoresis

Todd McDonald, Simon Sheng, Brian Stanley, Dawn Chen, Young Ko, Robert N. Cole, Peter Pedersen, Jennifer E. Van Eyk

Research output: Contribution to journalArticlepeer-review

Abstract

Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane sub-proteome as a model proteome, we compared for the first time the ability of three protein separation methods (two-dimensional liquid chromatography using the ProteomeLab™ PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one- and two-dimensional LC allowed for the separation of the α-subunit of F1 F0 ATP synthase that differed due to a change in pl or hydrophobicity.

Original languageEnglish (US)
Pages (from-to)2392-2411
Number of pages20
JournalMolecular and Cellular Proteomics
Volume5
Issue number12
DOIs
StatePublished - Dec 2006

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

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