Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Robinson, N.; Dale, Marsha P.; Talbert, Linda

doi:10.1016/0003-9861(90)90438-5

Cited by 15 publications

(9 citation statements)

References 13 publications

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“…Subunit analysis of the cytochrome bc\ complex by C18 reverse-phase HPLC required somewhat different incubation and gradient elution conditions than had previously been successful with bovine cytochrome c oxidase (Robinson et al, 1990; Y.-C. Liu, L. Sowdal, and N. C. Robinson, unpublished results). With cytochrome bc\, two relatively minor but important changes had to be made in the sample preparation and elution protocols: (i) during both sample preparation and gradient elution, the concentration of TFA had to be increased to 0.2%, and (ii) at least 5 mg of dodecyl maltoside per milliliter had to be present during the 45-60-min sample incubation in water containing 0.2% TFA.…”

Section: Resultsmentioning

confidence: 99%

“…Sample and Column Preparation. Cytochrome bc\ (50-700 Mg) was prepared for Qg reverse-phase HPLC analysis by a protocol that was similar to that used for subunit analysis of cytochrome c oxidase (Robinson et al, 1990). With cytochrome bc\, two minor but important changes had to be made in the procedure: (i) the final concentration of dodecyl maltoside in the sample must be at least 5 mg/mL; (ii) the sample must be incubated at room temperature for at least 45-60 min after acidification to 0.2% TFA.…”

Section: Methodsmentioning

confidence: 99%

“…NaDodSC>4-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Each HPLC peptide-containing fraction was analyzed for its subunit content by SDS-PAGE using 6-cm-long gels and either the Tricine buffer method of or the 15% acrylamide/SDS/2 M urea system described by Robinson et al (1990). The first method separates all 11 subunits and is particularly good at resolving the low molecular weight subunits, but the latter system gives better separation of the four largest molecular weight subunits.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, reverse-phase high-performance liquid chromatography (HPLC)1 has been successfully used to separate and quantitate the subunit content of bovine cytochrome c oxidase (Robinson et al, 1990; Y.-C. Liu, L. Sowdal, and N. C. Robinson, unpublished results).…”

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confidence: 99%

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Subunit Analysis of Bovine Cytochrome bc1 by Reverse-Phase HPLC and Determination of the Subunit Molecular Masses by Electrospray Ionization Mass Spectrometry

Musatov¹,

Robinson²

1994

Biochemistry

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A sensitive and simple scheme was developed for the rapid separation of mitochondrial complex III subunits by reverse-phase high-performance liquid chromatography (reverse-phase HPLC). Ten of the 11 subunits of cytochrome bc1 complex were separated with nearly baseline resolution between each peak. Cytochrome b was precipitated by acetonitrile on the column and could not be analyzed; the 10 other polypeptides were positively identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrospray ionization mass spectrometry (ESI/MS). The ESI/MS-determined molecular masses for subunits II, VI, VIII, IX, and XI are in excellent agreement with previously reported values; i.e., all are within +/- 2 mass units per 10 kDa. None of the other subunits gave molecular masses that agree with the published sequence values. The molecular mass of subunit I is 49 236 Da, which is far greater than the molecular mass of 35,833 Da calculated from the reported DNA sequence [Gencic et al. (1991) Eur. J. Biochem. 199, 122-131]. The Fe-S protein (subunit V) gives two masses which differ by 60 mass units, presumably due to either the partial loss of the two sulfur atoms or microheterogeneity. Neither mass agrees with the sequence value, the larger mass being 39 mass units lower than expected from the sequence. The molecular masses of subunits VII and X are 81 and 129 Da larger, respectively, than those calculated from their sequences [Borchart et al. (1986) FEBS Lett. 200, 81-86; Schägger et al. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 307-311].(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Subunit Analysis of Bovine Cytochrome bc1 by Reverse-Phase HPLC and Determination of the Subunit Molecular Masses by Electrospray Ionization Mass Spectrometry

Musatov¹,

Robinson²

1994

Biochemistry

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“…As studies focus on individual Complex I subunits, an analytical method for their separation and analysis is required. Our laboratory previously developed RP-HPLC methods for resolving all but the most hydrophobic, mitochondrially-encoded subunits of bovine heart cytochrome c oxidase [[20,21] and cytochrome bc 1 22]. Combined with mass spectrometry analysis of the resolved subunits, we have used this methodology to detect and quantify ROS-induced damage at selective sites within cytochrome c oxidase [23,24].…”

Section: Introductionmentioning

confidence: 99%

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

Lemma-Gray

Valušová

Carroll

et al. 2008

Analytical Biochemistry

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An effective method was developed for isolation and analysis of bovine heart Complex I subunits. The method utilizes C18 reversed-phase 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. Nine subunits do not elute including B14.7, MLRQ and the seven mitochondrial-encoded subunits. The method, with UV detection, is suitable for either analytical (< 50 µg protein) or preparative (>250 µg protein) applications. Subunit(s) eluting in each chromatographic peak were initially determined by MALDI-TOF/MS with subsequent positive identification by reversed-phase HPLC-ESI/MS/MS analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Scaffold analysis software combined with a X! Tandem protein search engine. The RP-HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity and ease of application.

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Novel methodology for topological studies of photosynthetic membrane protein complexes

Vater

Heinze²,

Friedrich³

et al. 1996

Ber Bunsenges Phys Chem

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Novel techniques have been developed for the investigation of photosynthetic membrane protein complexes from the thylakoid membrane of spinach. The protein components of photosystem I1 were fractionated by a solid phase extraction procedure using Chromabond cartridges. The cytochrome b6f complex was dissociated with high concentrations of the nonionic detergent n-dodecyl-b-D-maltoside. Subsequently the subunits of both membrane protein complexes were separated by high resolution reversed phase HPLC. Nearest neighbour relationships were investigated in the cytochrome b6f complex by molecular cross-linking with o-phthaldialdehyde. A methodology has been developed to identify the cross-linking sites on the molecular level by tryptic digestion of the protein conjugates and separation of the fragments by high resolution HPLC. Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS) was used as a sensitive tool to identify the protein components and crosslinked peptide fragments of photosystem I1 and the cytochrome b6f complex.

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Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Cited by 15 publications

References 13 publications

Subunit Analysis of Bovine Cytochrome bc1 by Reverse-Phase HPLC and Determination of the Subunit Molecular Masses by Electrospray Ionization Mass Spectrometry

Subunit Analysis of Bovine Cytochrome bc1 by Reverse-Phase HPLC and Determination of the Subunit Molecular Masses by Electrospray Ionization Mass Spectrometry

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

Novel methodology for topological studies of photosynthetic membrane protein complexes

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