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

Patrizia Lemma-Gray, Eva Valušová, Christopher A. Carroll, Susan T. Weintraub, Andrej Musatov, Neal C. Robinson

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

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 μg protein) or preparative (>250 μg 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.

Original languageEnglish (US)
Pages (from-to)116-121
Number of pages6
JournalAnalytical Biochemistry
Volume382
Issue number2
DOIs
StatePublished - Nov 15 2008

Keywords

  • ESI/MS/MS
  • Liquid chromatography
  • MALDI-TOF
  • Mitochondrial electron transport complexes
  • NADH dehydrogenase
  • Subunit analysis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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