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Integrating two chromatographies in a co-fractionation mass spectrometry approach improved protein-metabolite interactions (PMIs) mapping capabilities and revealed novel protein-ligand pairings in Escherichia coli.

ABSTRACT: The protein-metabolite interactome remains understudied. We previously developed a co-fractionation mass-spectrometry-based approach called PROMIS to address this gap. PROMIS relies on size separation chromatography of protein-metabolite complexes from lysates, followed by proteomics and metabolomics analysis of the obtained fractions. Co-elution of metabolites and proteins is used to infer interactions. However, in a single separation, a metabolite cofractionates with hundreds of proteins, of which one or a few are 'true interactors,' whereas all others represent coincidental co-elution. Identifying the true binders represents the most significant challenge in using CF-MS-based methods for mapping PMIs. To address this challenge, we decided to integrate two chromatographies, size exclusion, and ion exchange, to improve PMI mapping capabilities and generate a PMI network in a model bacteria Escherichia coli. Although many complexes will have a similar size or charge, only a few should share both characteristics. The resulting interactome, comprising 994 interactions between 51 metabolites and 465 proteins, is enriched for known and predicted interactions, supporting the starting premise of this work that integrating different separation methods can assist in constructing protein-metabolite interactomes. Analysis of the protein targets predicted for the different metabolites revealed novel functional insights, such as the association between proteinogenic dipeptides and fatty acid metabolism. Moreover, we identified a novel, inhibitory interaction between riboflavin degradation product lumichrome and de novo pyrimidine synthesis enzyme, orotate phosphoribosyltransferase (pyrE). Lumichrome supplementation inhibited biofilm formation, on par with the known phenotype of ΔpyrE mutant strain. Based on published and our data, we hypothesize that lumichrome functions to integrate pyrimidine and riboflavin metabolism with quorum sensing and biofilm formation.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse phase, Liquid Chromatography MS - positive - reverse phase

PROVIDER: MTBLS9098 | MetaboLights | 2025-04-25

REPOSITORIES: MetaboLights

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Project description:Many proteins form stable complexes that coordinate diverse and complex cellular processes. Systematic analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. Protein complexes are usually identified using either yeast-two-hybrid (Y2H) or affinity purification-mass spectrometry (AP-MS); however, these methods are low throughput and require genetic manipulation. While there have been advances in techniques for proteome-wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatographic (SEC) with label-free quantitative liquid chromatography tandem-MS (LC-MS/MS) profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme (GBM) cells. We identified 1794 proteins that overlapped between two biological replicates, of which 1244 proteins were characterized as existing within stably associated putative complexes. We demonstrated a high degree of SEC reproducibility by comparing protein elution profiles between the two independent fractionation procedures, and co-elution of several chromatin remodeling and DNA damage repair complexes was confirmed by independent co-immunoprecipitation (co-IP) and immunoblot analyses. Co-IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins, and between HDAC1 and CHD4. HDAC1/2 also co-migrated with various SIN3a and NuRD components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co-elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC-MS analysis for system-wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.

			Action	DRS
	IEXC_R1_10_neg.raw	Raw
	IEXC_R1_10_pos.raw	Raw
	IEXC_R1_11_neg.raw	Raw
	IEXC_R1_11_pos.raw	Raw
	IEXC_R1_12_neg.raw	Raw

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Integrating two chromatographies in a co-fractionation mass spectrometry approach improved protein-metabolite interactions (PMIs) mapping capabilities and revealed novel protein-ligand pairings in Escherichia coli.

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