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Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

ABSTRACT: Recently, several mass spectrometry methods have utilized protein structural stability for the quantitative study of protein-ligand engagement. These protein-denaturation approaches, which include thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX), evaluate ligand-induced denaturation susceptibility changes with a MS-based readout. The different techniques of bottom-up protein-denaturation methods each have their own advantages and challenges. Here, we report the combination of protein-denaturation principles with quantitative cross-linking mass spectrometry using isobaric quantitative protein interaction reporter technologies. This method enables the evaluation of ligand-induced protein engagement through analysis of cross-link relative ratios across chemical denaturation. As a proof of concept, we found ligand-stabilized cross-linked lysine pairs in well-studied bovine serum albumin and ligand bilirubin. These links map to the known binding sites Sudlow Site I and subdomain IB. We propose that protein denaturation and qXL-MS can be combined with similar peptide-level quantification approaches, like SPROX, to increase the coverage information profiled for facilitating protein-ligand engagement efforts.

SUBMITTER: Mathay M

PROVIDER: S-EPMC10848897 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

Mathay Martin M Keller Andrew A Bruce James E JE

Analytical chemistry 20230612 25

Recently, several mass spectrometry methods have utilized protein structural stability for the quantitative study of protein-ligand engagement. These protein-denaturation approaches, which include thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX), evaluate ligand-induced denaturation susceptibility changes with a MS-based readout. The different techniques of bottom-up protein-denaturation methods each have their own advantages and challenges. Here, we rep ...[more]

PMID: 37307416

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Project description:Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

Dataset Information

Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

Publications

Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

Similar Datasets

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