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Detection of Complex I and II in BN-PAGE gels

ABSTRACT: Study of the fully assembled complex I and complex II in mitochondria of oocytes.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Xenopus Laevis (african Clawed Frog) Mus Musculus (mouse)

TISSUE(S): Skeletal Muscle, Oocyte

SUBMITTER: Aida Rodriguez-Nuevo

LAB HEAD: Elvan Boke

PROVIDER: PXD025371 | Pride | 2022-05-09

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2020MG007_AIRO_001_01_45pto.pdResult	Other
	2020MG007_AIRO_001_01_45pto.raw	Raw
	2020MG007_AIRO_002_01_45pto.pdResult	Other
	2020MG007_AIRO_002_01_45pto.raw	Raw
	2020MG007_AIRO_003_01_45pto.pdResult	Other

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1 - 5 of 17

Publications

Oocytes maintain ROS-free mitochondrial metabolism by suppressing complex I.

Rodríguez-Nuevo Aida A Torres-Sanchez Ariadna A Duran Juan M JM De Guirior Cristian C Martínez-Zamora Maria Angeles MA Böke Elvan E

Nature 20220720 7920

Oocytes form before birth and remain viable for several decades before fertilization1. Although poor oocyte quality accounts for most female fertility problems, little is known about how oocytes maintain cellular fitness, or why their quality eventually declines with age2. Reactive oxygen species (ROS) produced as by-products of mitochondrial activity are associated with lower rates of fertilization and embryo survival3-5. Yet, how healthy oocytes balance essenti ...[more]

PMID: 35859172

Similar Datasets

Shotgun Proteomics of BN-PAGE-Resolved Chloroplasts

Project description:Blue Native-PAGE resolves protein complexes in their native state. When coupled with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large-scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of wild-type chloroplasts into 6 bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight resolution. Significantly, our approach achieves a depth of identification comparable to traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to our ‘mass western’ approach to BN-PAGE. The coupling of BN-PAGE to MS/MS allows for a large-scale comparison of protein complexes between samples and the identification of proteins in high molecular weight complexes. In parallel we have analyzed chloroplasts from a leaf reticulate mutant (re-6) to demonstrate the possibility for comparative analyses. Our results provide a useful resource for the chloroplast community and this strategy is anticipated to be widely adaptable to other sub-cellular compartments.

2016-12-23 | PXD003162 | Pride

ETFDH comigrates with CIII in BN-PAGE

Project description:Proteomic analysis of 13 bands from BN-PAGE immunostaining of mouse skeletal muscle mitochondrial proteins: Coenzyme Q (Q) is a key lipid electron transporter, but several aspects of its biosynthesis and redox homeostasis remain undefined. Various flavoproteins reduce the Q; however, in eukaryotes, only the OXPHOS-complex III (CIII) oxidizes the QH2. The mechanism of action of CIII is still debated. We demonstrate that the Q-reductase ETFDH is essential for CIII activity in skeletal muscle. We identify a complex involving ETFDH, CIII, and the Q-biosynthesis regulator COQ2 that directs electrons from lipid substrates to the respiratory chain, reducing electron leak and ROS production. This metabolon maintains Q levels, minimizes QH2-reductive stress, and improves OXPHOS efficiency. Muscle-specific ETFDH-/- mice develop myopathy due to CIII dysfunction, indicating that ETFDH is a required OXPHOS component and a potential therapeutic target for mitochondrial redox medicine

2023-10-24 | PXD045352 | Pride

Proteomics profiling of Lrpprc knockout mice liver mitochondria following resolution by hybrid CN/BN PAGE

Project description:Mouse models of genetic mitochondrial diseases are generally used to understand specific molecular defects and their biochemical consequences, but rarely to map compensatory changes allowing survival. Here we took advantage of the extraordinary mitochondrial resilience of hepatic Lrpprc knockout mice to explore this question using native proteomics profiling and lipidomics. In these mice, lack of the mtRNA binding protein LRPPRC induces a global mitochondrial translation defect and a severe reduction (>80%) in the assembly and activity of the electron transport chain (ETC) complex IV (CIV). Yet, animals show no signs of liver failure and capacity of the ETC is completely preserved. Beyond stimulation of mitochondrial biogenesis, results show that the abundance of mitoribosomes per unit of mitochondria is increased and proteostatic mechanisms are induced in absence of LRPPRC to preserve a balance in the availability of mitochondrial- vs nuclear-encoded ETC subunits. At the level of individual organelles, a strong preferential integration of residual CIV in supercomplexes (SCs) is observed, pointing to a role of these supramolecular arrangements in preserving ETC function. This can be mechanistically explained by the upregulation of the assembly factor COX7A2L and its stabilization into SCs. Furthermore, lipidomics and proteomics evidences indicate a shift in the phospholipids composition of mitochondrial membrane including an upregulation of SC stabilizing cardiolipin (CL) species, and several CL-binding protein complexes playing key roles in CL metabolism. Together these data reveal a complex in vivo network of molecular adjustments involved in preserving mitochondrial integrity in energy consuming organs facing OXPHOS defects, which could be therapeutically exploited.

2021-09-09 | PXD021867 | Pride

Plastoglobules BN-PAGE experiment (Arabidopsis PGs under unstress and high light conditions)

Project description:Proteome comparison of isolated plastoglobules (PGs) from Arabidopsis thaliana (Col-0 ecotype) under unstress (UNS) and high light (HL) stress conditions. For both conditions, PGs were isolated from three independent set of plants each (i.e. three for UNS and three for HL). PGs were isolated from the plants when they were 4 weeks old under long day photoperiod (16 hours of light and 8 darkness). The light stress was applied to the designated set of plants 5 days prior the completion of the 4 weeks by shifting the light intensity from 120 uE to 500 E. The PGs were solubilized with 1.1 % n-Dodecyl--D-Maltoside and separated in a Blue-Native gel. After the electrophoretic separation, gel lanes were cut and further divided into 10 independent slices corresponding to different molecular weights. These gel slices were subjeted to in-gel digestion with trypsin and the resulting peptides labeled with TMT reagents. Labeled peptides were mixed 1:1 and purified by solid phase extraction using c18 StageTips. Eluted peptides were sprayed into a ThermoScientific Q-Exactive HF-X mass spectrometer. Survey scans were taken in the Orbi trap (120000 resolution, determined at m/z 200) and the top 20 ions in each survey scan are then subjected to automatic higher energy collision induced dissociation (HCD) with fragment spectra acquired at 45000 resolution. Additionally, PG samples that were not separated in a Blue-native gel were trypsin digested and subjected for mass-spectrometry analysis as previously described.

2021-04-13 | PXD019735 | Pride

Combining BN-PAGE and microscopy techniques to investigate pigment-protein complexes and plastid transitions in citrus fruit.

Project description:BackgroundChlorophyll and carotenoids, the most widely distributed lipophilic pigments in plants, contribute to fruit coloration during development and ripening. These pigments are assembled with pigment-protein complexes localized at plastid membrane. Pigment-protein complexes are essential for multiple cellular processes, however, their identity and composition in fruit have yet to be characterized.ResultsBy using BN-PAGE technique in combination with microscopy, we studied pigment-protein complexes and plastid transformation in the purified plastids from the exocarp of citrus fruit. The discontinuous sucrose gradient centrifugation was used to isolate total plastids from kumquat fruit, and the purity of isolated plastids was assessed by microscopy observation and western blot analysis. The isolated plastids at different coloring stages were subjected to pigment autofluorescence observation, western blot, two-dimensional electrophoresis analysis and BN-PAGE assessment. Our results demonstrated that (i) chloroplasts differentiate into chromoplasts during fruit coloring, and this differentiation is accompanied with a decrease in the chlorophyll/carotenoid ratio; (ii) BN-PAGE analysis reveals the profiles of macromolecular protein complexes among different types of plastids in citrus fruit; and (iii) the degradation rate of chlorophyll-protein complexes varies during the transition from chloroplasts to chromoplasts, with the stability generally following the order of LHCII > PS II core > LHC I > PS I core.ConclusionsOur optimized methods for both plastid separation and BN-PAGE assessment provide an opportunity for developing a better understanding of pigment-protein complexes and plastid transitions in plant fruit. These attempts also have the potential for expanding our knowledge on the sub-cellular level synchronism of protein changes and pigment metabolism during the transition from chloroplasts to chromoplasts.

| S-EPMC9675244 | biostudies-literature

Membrane Fraction Complexome of Nitrososphaera viennensis with BN-PAGE and Examination of the Archaeal Ammonia Monooxygenase Complex via SDS-Tricine-PAGE

Project description:Information on the files related to the respective experiments can be found in the metadata file titled “Metadata_for_Nviennensis_Experiments.xlsx”. The data found here encompasses three related mass spectrometry experiments: 1.) BN-PAGE: Biomass of Nitrososphaera viennensis grown in a bioreactor was lysed and membrane fractions were harvested using an ultracentrifuge. Membrane proteins were extracted using n-dodecyl-beta-D-maltoside (DDM) and loaded on a 3-12% gradient blue native PAGE gel. Selected bands were cut out and analyzed via mass spectrometry. 2.) SDS-Tricine-PAGE of AMO Band: A band containing a large amount of the ammonia monooxygenase complex (AMO) was denatured and run on a 15% SDS-Tricine-PAGE gel to identify individual subunits. 3.) AmoC from SDS-Tricine-PAGE: A band containing a high amount of AmoC from the SDS-Tricine-PAGE gel was processed with three separate digestive enzymes (trypsin, chymotrypsin, or GluC) to identify unique peptides among the six AmoC homologs found in the genome of N. viennensis.

2023-02-13 | PXD035349 | Pride

Isolation of thylakoid membrane complexes from rice by a new double-strips BN/SDS-PAGE and bioinformatics prediction of stromal ridge subunits interaction.

Project description:Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new "Double-Strips BN/SDS-PAGE" method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.

| S-EPMC3102703 | biostudies-literature

Comparison of human primordial oocyte follicles to ovarian somatic cells

Project description:Analysis primordial follicle compared to ovarian somatic cells proteome in humans.

2022-05-09 | PXD025369 | Pride

Proteomic profile of human breast cancer cells expressing normal levels or absent of the long non-coding RNA NORAD.

Project description:We describe here the proteome of the human breast cancer cell line MDA-MB-231 expressing normal levels of the lncRNA NORAD, or after knock-down NORAD with siRNA and LNA gapmers.

2023-08-28 | PXD039920 | Pride

Cross-linked ammonia monooxygenase complex in BN-PAGE gel from Nitrososphaera viennensis

Project description:A band containing the ammonia monooxygenase complex from Nitrosospheara viennensis was cut from a Blue Native PAGE gel and cross linked using DSSO.

2023-02-13 | PXD034475 | Pride

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