Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

Anal Biochem. 2008 Nov 15;382(2):116-21. doi: 10.1016/j.ab.2008.07.029. Epub 2008 Jul 31.


An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 microg protein) or preparative (>250 microg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Chromatography, High Pressure Liquid / methods*
  • Electron Transport Complex I / analysis*
  • Electron Transport Complex I / metabolism
  • Mass Spectrometry*
  • Mitochondria, Heart / chemistry*
  • Protein Subunits / analysis
  • Protein Subunits / metabolism
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
  • Tandem Mass Spectrometry


  • Protein Subunits
  • Electron Transport Complex I