Dataset Information

Multiplexed isobaric quantitative Protein Interaction Reporter (iqPIR) cross-linking on standard proteins

ABSTRACT: Initial demonstration of multiplexed isobaric quantitative Protein Interaction Reporter technology using two multiplexed samples consisting of 5 cross-linked standard proteins including alcohol dehydrogenase 1 (ADH1_YEAST), albumin (ALBU_BOVIN), cytochrome c (CYC_BOVIN), hemoglobin (HBA_HUMAN), and myoglobin (MYG_HORSE).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Equus Caballus (horse) Homo Sapiens (human) Bos Taurus (bovine) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Juan Chavez

LAB HEAD: James E. Bruce

PROVIDER: PXD026037 | Pride | 2023-03-11

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	111820_6plex_protein_mix_sample_1_1.mzXML	Mzxml
	111820_6plex_protein_mix_sample_1_1.raw	Raw
	111829_6plex_protein_mix_sample_2_1.mzXML	Mzxml
	111829_6plex_protein_mix_sample_2_1.raw	Raw
	iprophet-xl.pep.xml	Pepxml

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Publications

Multiplexed Cross-Linking with Isobaric Quantitative Protein Interaction Reporter Technology.

Chavez Juan D JD Keller Andrew A Wippel Helisa H HH Mohr Jared P JP Bruce James E JE

Analytical chemistry 20211209 50

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful technique for interrogating protein structures and interactions. When combined with quantitative proteomics strategies, protein conformational and interaction dynamics can be probed. Quantitative XL-MS has been demonstrated with the use of stable isotopes incorporated metabolically or into the cross-linker molecules. Isotope-labeled cross-linkers have primarily utilized deuterium and rely on MS1-based quantitation of p ...[more]

PMID: 34882395

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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.