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.
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.
Lemma-Gray
Patrizia
P
Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA.
Valusová
Eva
E
Carroll
Christopher A
CA
Weintraub
Susan T
ST
Musatov
Andrej
A
Robinson
Neal C
NC
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D042967:Electron Transport Complex I / Q000032:analysis* / Q000378:metabolism; D013058:Mass Spectrometry; D008929:Mitochondria, Heart / Q000737:chemistry*; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry
D021122:Protein Subunits; D042967:Electron Transport Complex I