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Cdc48A pulldown from an amino lonk column baited with SAMP1

ABSTRACT: H. volcanii Cdc48a, RecJ3/4 and RNase J associate with Ubl-coated beads. A) Strategy to isolate H. volcanii proteins that bind agarose beads charged with monomeric Ubl (vs. BSA) as bait. Cell lysate was from triplicate cultures of H. volcanii NH02-pJAM957. B) Left, Non-reducing SDS-PAGE of H. volcanii proteins that bound BSA- (control, lane 1) vs. Ubl- (lane 2) decorated beads in the presence of ATP. Gels were stained with SYPRO Ruby. Red bar, HMW(250kDa) and LMW(50kDa) (high and low molecular weight) regions of gel excised from lanes 1 and 2 for LC- MS/MS analysis. Right, Proteins identified at > 99.9% probability and < 0.1 % false discovery rate (FDR) in the Ubl (not BSA) samples. Similar trend was observed in independent experiment. ATP was required for this association. Theor. Mr, theoretical molecular mass based on genome sequence. Normalized total spectra of sample minus control is indicated.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Haloferax Volcanii Ds2

SUBMITTER: Swathi Dantuluri

LAB HEAD: Julie Maupin Furlow

PROVIDER: PXD019897 | Pride | 2023-06-22

REPOSITORIES: Pride

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Project description:Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases. While it is clear that cell lines derived from different cell types have differential proteome levels, increased understanding of large-scale functional differences requires additional information beyond abundance level measurements, including how protein conformations and interactions are altered in certain cell types to shape functional landscapes. Here, we employed quantitative in vivo protein cross-linking coupled to mass spectrometry to probe large-scale protein conformational and interaction changes among three commonly employed human cell lines, HEK293, MCF-7, and HeLa cells. Isobaric quantitative Protein Interaction Reporter (iqPIR) technologies were used to obtain quantitative values of cross-linked peptides across three cell lines. These data illustrated highly reproducible (R2 values larger than 0.8 for all biological replicates) quantitative interactome levels across multiple biological replicates. We also measured protein abundance levels in these cells using data independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteomics information allowed visualization of cell type-specific interactome changes mediated by proteome level adaptations as well as independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the biggest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study systems-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight on cellular functional landscapes.

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	50.RAW__F007089_.mzid.gz	Mzid

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Cdc48A pulldown from an amino lonk column baited with SAMP1

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