Project description:EndoMAP aims to systematically dissect the human endosomal protein complexes. To purify endosomes from human cells, a modified Endo-IP protocol was developed to be compatible with complexomics methodologies and evaluated by DIA-MS. Three independent biological replicates of human endosomes purified by the modified Endo-IP protocol were subjected to Blue Native PolyAcrylamide Gel Electrophoresis (BN-PAGE) co-fractionation to identify possible protein complexes based on correlation profiling.
Project description:The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, we provides evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes. When the affinity-isolated GlyR complexes were fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β-GPHN appeared as the most abundant complex with a molecular weight of approximately 1 MDa, and GlyR α1β-GPHN-IQSEC3 as a minor protein complex of approximately 1.2 MDa. A third GlyR α1β-GPHN-IQSEC2 complex existed at the lowest amount with a mass similar to the IQSEC3-containing complex. Using yeast two-hybrid we demonstrate that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. Our data provide direct evidence of the interaction of IQSEC3 with GlyR-GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.
Project description:[original Title] Transcriptomic responses to heat-stress in invasive and native blue mussels (genus Mytilus): molecular correlates of invasive success. Invasive species are increasingly prevalent in marine ecosystems worldwide. Although many studies have examined the ecological effects of invasives, little is known about physiological mechanisms that might contribute to invasive success. The mussel Mytilus galloprovincialis, a native of the Mediterranean Sea, is a successful invader on the central and southern coasts of California, where it has largely displaced the native congener, Mytilus trossulus. It has been previously shown that thermal responses of several physiological traits may underlie the capacity of M. galloprovincialis to out-compete M. trossulus in warm habitats. To elucidate possible differences in stress-induced gene expression between these congeners, we developed an oligonucleotide microarray with 8,874 probes representing 4,488 different genes that recognized mRNAs of both species. In acute heat-stress experiments, 1,531 of these genes showed temperature-dependent changes in gene expression that were highly similar in the two congeners. In contrast, 96 genes showed species-specific responses to heat-stress, functionally characterized by their involvement in oxidative stress, proteolysis, energy metabolism, ion transport, cell signaling, and cytoskeletal reorganization. The gene that showed the biggest difference between the species was the gene for the molecular chaperone small heat shock protein 24, which was highly induced in M. galloprovincialis and showed only a small change in M. trossulus. These different responses to acute heat-stress may help to explain—and predict—the invasive success of M. galloprovincialis in a warming world.
Project description:Here, we demonstrate a pilot development of multi-dimensional native proteomics technology for protein complex identification and topology characterization (Mud-NatPIT) in complex biological samples via coupling native size exclusion chromatography (SEC), online native capillary electrophoresis-mass spectrometry (nCE-MS), surface-induced dissociation (SID), and multi-level proteomics. The Mud-NatPIT enabled the detection of 124 unique native endogenous protein complexes with masses up to 1.5 MDa from a whole cell lysate, identified 46 unique protein complexes in a discovery mode, and provided topological information for 39 protein complexes. The Mud-NatPIT is broadly applicable for characterizing protein complexes in cells, tissues, and biological fluids.
Project description:GABAA receptors are the major inhibitory receptors in the brain. They are hetero-pentamers with a composition of predominantly two α, two β and one γ or δ subunit. From the six α subunit genes, the α5 subunit displays a limited spatial expression pattern and is known to mediate both phasic and tonic inhibition. In this study, using immunoaffinity-based proteomics we identified the α5 subunit containing receptor complexes in hippocampus and olfactory bulb. The α1-α5 interaction was identified in both brain regions albeit with significantly different stoichiometries. In line with this, reverse IPs using anti-α1 antibody showed the α5-α1 co-occurrence and validated the quantitative difference. In addition, we showed that the association of Neuroligin 2 with α1-containing receptors was much higher in olfactory bulb than hippocampus, which was confirmed using blue native gel electrophoresis and quantitative mass spectrometry. Finally, immunocytochemical staining revealed co-localization of α1 and α5 subunits in post-synaptic puncta in the hippocampus.
Project description:In this dataset, we analyzed Aquaporin Z using native mass spectrometry. The data was then input into ProSight Native to determine the protein complex composition.