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

Musatov, Andrej; Robinson, N.

doi:10.1021/bi00201a001

Cited by 28 publications

(22 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cyt f was eluted at a concentration of 45% acetonitrile. The Rieske protein could not be detected, while cyt b6 was not released from the column as was also observed for the cyt bc, complex [15]. Being the most hydrophobic protein of the complex it either was partially precipitated prior to HPLC or retained on the column.…”

Section: Resultsmentioning

confidence: 63%

Novel methodology for topological studies of photosynthetic membrane protein complexes

Vater

Heinze²,

Friedrich³

et al. 1996

Ber Bunsenges Phys Chem

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 63%

Novel methodology for topological studies of photosynthetic membrane protein complexes

Vater

Heinze²,

Friedrich³

et al. 1996

Ber Bunsenges Phys Chem

View full text Add to dashboard Cite

show abstract

“…Many of the difficulties arise from their hydrophobicity and from the associated lack of procedures for purifying membrane proteins in a suitable form for mass spectrometric analysis. Usually, detergents are used to extract and purify membrane proteins, but they are incompatible with the protein ionization methods used in MS, and so they have to be removed from the purified protein (often by chromatography in organic solvents) before analysis can be undertaken (1)(2)(3). An alternative approach is to extract and fractionate the membrane proteins directly in organic solvents (3)(4)(5).…”

mentioning

confidence: 99%

Identification of membrane proteins by tandem mass spectrometry of protein ions

Carroll

Altman

Fearnley

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The most common way of identifying proteins in proteomic analyses is to use short segments of sequence (''tags'') determined by mass spectrometric analysis of proteolytic fragments. The approach is effective with globular proteins and with membrane proteins with significant polar segments between membranespanning ␣-helices, but it is ineffective with other hydrophobic proteins where protease cleavage sites are either infrequent or absent. By developing methods to purify hydrophobic proteins in organic solvents and by fragmenting ions of these proteins by collision induced dissociation with argon, we have shown that partial sequences of many membrane proteins can be deduced easily by manual inspection. The spectra from small proteolipids (1-4 transmembrane ␣-helices) are dominated usually by fragment ions arising from internal amide cleavages, from which internal sequences can be obtained, whereas the spectra from larger membrane proteins (5-18 transmembrane ␣-helices) often contain fragment ions from N-and/or C-terminal parts yielding sequences in those regions. With these techniques, we have, for example, identified an abundant protein of unknown function from inner membranes of mitochondria that to our knowledge has escaped detection in proteomic studies, and we have produced sequences from 10 of 13 proteins encoded in mitochondrial DNA. They include the ND6 subunit of complex I, the last of its 45 subunits to be analyzed. The procedures have the potential to be developed further, for example by using newly introduced methods for protein ion dissociation to induce fragmentation of internal regions of large membrane proteins, which may remain partially folded in the gas phase.mitochondria ͉ complex I ͉ ND6 subunit ͉ membrane proteome ͉ proteolipids O ne-third of proteins encoded in genomes are hydrophobic membrane proteins, and their analysis by mass spectrometric methods is more difficult than the analysis of hydrophilic proteins. Many of the difficulties arise from their hydrophobicity and from the associated lack of procedures for purifying membrane proteins in a suitable form for mass spectrometric analysis. Usually, detergents are used to extract and purify membrane proteins, but they are incompatible with the protein ionization methods used in MS, and so they have to be removed from the purified protein (often by chromatography in organic solvents) before analysis can be undertaken (1-3). An alternative approach is to extract and fractionate the membrane proteins directly in organic solvents (3-5).The measurement of the mass of a protein allows the presence but not the location of any posttranslational modifications to be detected, but it does not provide a reliable means of identifying a protein in sequence data-bases, as required in proteomic analyses. Usually, short segments of protein sequence (sequence tags), obtained by mass spectrometric analysis of proteolytic peptides, are used for this purpose (6). This method provides a highly effective means of generating partial sequences from globular protei...

show abstract

“…Based on the ubiquinone content of the samples employed, we simply assume that maximally 36% QCR in the purified preparation contains a full set of redox centers; that is 1 mol each of Q 10 -(2), heme b L -(1), heme b H -(1), heme c 1 - (1), and ISP- (1). In parentheses the maximal number of reducing equivalent that can be retained is given.…”

Section: Reaction Of Fully Reduced Qcr With Dioxygen In the Presence mentioning

confidence: 99%

“…Furthermore, we assume some constraints as follows. (i) QCR during the reaction is a closed system that interacts with cytochrome c of the oxidizing system only through heme c 1 In this reaction scheme (Fig. 5) the fully reduced QCR is at C1R1 if we regard this arrangement as a (sparse) matrix and if the location is represented by a combination of the column (C) and the row (R).…”

Section: Oxidation Of Partially Reduced Qcr-mentioning

confidence: 99%

“…Ubiquinol-cytochrome c oxidoreductase (QCR 1 ; bc 1 complex or complex III) is one of the three sites for energy conservation in the mitochondrial respiratory chain. This mitochondrial enzyme consisting of 11 subunits (1) translocates protons from the matrix side of mitochondrial inner membranes toward the cytoplasmic side coupled with electron transfer from ubiquinol to cytochrome c (2,3).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Oxidation Process of Bovine Heart Ubiquinol-Cytochrome c Reductase as Studied by Stopped-flow Rapid-scan Spectrophotometry and Simulations Based on the Mechanistic Q Cycle Model

Orii

Miki²

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

Stopped-flow rapid-scan spectrophotometry was employed to study complicated oxidation processes of ubiquinol-cytochrome c reductase (QCR) that was purified from bovine heart mitochondria and maximally contained 0.36 mol of ubiquinone-10/mol of heme c 1 . When fully reduced QCR was allowed to react with dioxygen in the presence of cytochrome c plus cytochrome c oxidase, the oxidation of b-type hemes accompanied an initial lag, apparently low potential heme b L was oxidized first, followed by high potential heme b H . Antimycin A inhibited the oxidation of both b-type hemes. The oxidation of heme c 1 was triphasic and became biphasic in the presence of antimycin A. On the other hand, starting from partially reduced QCR that was poised at a higher redox potential with succinate and succinatecytochrome c reductase, the b-type hemes were oxidized immediately without a lag. When the ubiquinone content in QCR was as low as 0.1 mol/mol heme c 1 the oxidation of the b-type hemes was almost suppressed. As the Q-deficient QCR was supplemented with ubiquinol-2, the rapid oxidation of b-type hemes was restored to some extent. These results indicate that a limited amount of ubiquinone-10 found in purified preparations of QCR is obligatory for electron transfer from the btype hemes to iron-sulfur protein (ISP) and heme c 1 .The

show abstract

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

Cited by 28 publications

References 24 publications

Novel methodology for topological studies of photosynthetic membrane protein complexes

Novel methodology for topological studies of photosynthetic membrane protein complexes

Identification of membrane proteins by tandem mass spectrometry of protein ions

Oxidation Process of Bovine Heart Ubiquinol-Cytochrome c Reductase as Studied by Stopped-flow Rapid-scan Spectrophotometry and Simulations Based on the Mechanistic Q Cycle Model

Contact Info

Product

Resources

About