Project description: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.
Project description:The majority of proteins are organized in protein complexes. Protein complexes represent an important cellular organizational layer, which regulate and catalyze most of the cellular processes. Within the field of proteomics several techniques such as Affinity Purification (AP) and co-fractionation (co-Frac) coupled to mass spectrometry (MS) were developed in order to study protein complexes. Our approach, deep interactome profiling coupled to MS (DIP-MS), is a combination of the benefits of these approaches by combining the selectivity of AP-MS together with a blue native-page size-based fractionation, in order to deconvolute the co-purified complexes, in which the bait is a constitutive subunit. To ensure high-quality quantitation along the fractionation gradient, and to keep data acquisition time limited, we developed a high-throughput (HT) sample preparation method and high-throughput data independent acquisition (DIA) method, enabling us to acquire almost one co-Frac gradient per day. Additionally, a tailored machine learning approach was devised – protein-protein interaction prophet (PPIprophet) which enabled the scoring of the co-elution proteins to retrieve PPIs respectively protein complexes. The method was employed to depict the complex modularity within the prefoldin and prefoldin-like protein complexes, allowing us to I) describe protein complex isoforms, II) derive stoichiometries and abundance distributions of co-purified complexes and III) identify reported and new client proteins and client complexes of the PAQosome, a multi-subunit co-chaperone complex, necessary for the stabilization and formation of multiple complexes such as the RNA polymerases (RNA Pol I, RNA Pol II, RNA Pol III). This study thereby demonstrates the applicability of our method and shows it strength and sensitivity by depicting the prefoldin complexes within only a single experiment.
Project description:Our current research focuses on the complex-centric quantification of pTyr protein complexes. We initially constructed a tri-functional probe based on pTyr superbinder (SH2-S) for the enrichment of pTyr protein complexes. The pTyr protein complexes were separated by ion exchange chromatography (IEC). To further quantify the complexes, we developed an algorithm for co-fractionation/mass spectrometry (CF/MS) protein complex screening and quantification.