TY - JOUR
T1 - 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
AU - Lemma-Gray, Patrizia
AU - Valušová, Eva
AU - Carroll, Christopher A.
AU - Weintraub, Susan T.
AU - Musatov, Andrej
AU - Robinson, Neal C.
N1 - Funding Information:
We thank Tiffany McDonald-Marsh and Linda Sowdal for their technical assistance in this project. Mass spectrometry analyses were performed in the UTHSCSA Institutional Mass Spectrometry Laboratory. This work was supported by grants from the National Institutes of Health (GMS 24795), the Robert A. Welch Foundation (AQ1481), and the University of Texas Health Science Center, San Antonio (VPN 125644).
PY - 2008/11/15
Y1 - 2008/11/15
N2 - 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.
AB - 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.
KW - ESI/MS/MS
KW - Liquid chromatography
KW - MALDI-TOF
KW - Mitochondrial electron transport complexes
KW - NADH dehydrogenase
KW - Subunit analysis
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U2 - 10.1016/j.ab.2008.07.029
DO - 10.1016/j.ab.2008.07.029
M3 - Article
C2 - 18721790
AN - SCOPUS:52049115385
VL - 382
SP - 116
EP - 121
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -