Dataset Information

Proximal Biotinylation Based Combinatory Approach for Isolating Plasma Membrane Proteins

ABSTRACT: Comprehensive profiling of the cell surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here, we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra in vitro labeling step that we called plasma membrane-BioID. The lipid modified BirA* enzyme (MyrPalm BirA*) was targeted to the plasma membrane where it effectively biotinylates plasma membrane proteins. Biotinylated proteins were then affinity purified and analyzed by mass spectrometry. Our analysis demonstrates that combining conventional sucrose density gradient centrifugation and plasma membrane-BioID is ideal to overcome the inherent limitations of identification of integral membrane proteins and yields highly pure plasma components for downstream proteomic analysis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Altuğ Kamacıoğlu

LAB HEAD: Nurhan Ozlu

PROVIDER: PXD018383 | Pride | 2020-08-28

REPOSITORIES: Pride

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Publications

Proximal Biotinylation-Based Combinatory Approach for Isolating Integral Plasma Membrane Proteins.

Akdag Mehmet M Yunt Zeynep Sabahat ZS Kamacioglu Altug A Qureshi Mohammed Haroon MH Akarlar Busra A BA Ozlu Nurhan N

Journal of proteome research 20200615 8

Comprehensive profiling of the cell-surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra <i>in vitro</i> labeling step, which we call Plasma Membrane-BioID. The lipid-modified BirA* enzyme (MyrPalm BirA*) was targeted to the inner leaflet of the plasma membrane, where it ...[more]

PMID: 32500712

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Project description:Biological CO2 mitigation by photosynthetic microorganisms has emerged as a promising approach for generating biomass-based energy during the course of CO2 fixation. Additionally, cyanobacteria-based biofuels have also garnered immense attention lately because of the depleting fossil fuel reserves and the growing energy demands. Synechococcus elongatus PCC 11801, a fast-growing, high CO2 tolerant, novel isolate of cyanobacteria, is an interesting candidate for metabolic engineering applications. Since under laboratory conditions, it exhibited a high level of tolerance to different environmental stresses (CO2, light, temperature, salts and butanol), it seemed like an encouraging prospect for the production of fuels and other industrially relevant chemicals. This is the first-ever functional global proteomics investigation of Synechococcus elongatus PCC 11801 isolate grown at elevated CO2 levels using high-throughput iTRAQ approach. Three independent biological replicates were analyzed and a total of 861 proteins were identified, out of which 492 proteins were found to be present in all the three replicates. Of these, 248 proteins showing the same trend across the three replicates (≥1.5 fold up-regulation or ≤0.66 fold down-regulation) were chosen for pathway analysis. The metabolic responses were marked with a down-regulation of inorganic carbon transporters alongside an induction of nitrogen transport and absorption proteins in order to uphold the apposite carbon-nitrogen equilibrium. Further acclimation progression exposed an increased expression of proteins taking part in photosynthesis and generation of light harvesting pigments such as chlorophyll. Similarly, a downshift of proteins involved in photoprotection and defense against ROS (reactive oxygen species) was observed. Another principal discovery was the perturbation in expression of proteins belonging to central metabolic pathways like glycolysis, TCA cycle, pentose phosphate pathway as a coping mechanism to high CO2 stress. Further, validation studies were carried out using MRM assays and western blotting of key altered proteins.

Dataset Information

Proximal Biotinylation Based Combinatory Approach for Isolating Plasma Membrane Proteins

Publications

Proximal Biotinylation-Based Combinatory Approach for Isolating Integral Plasma Membrane Proteins.

Similar Datasets

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