Multiplexed interactome analysis reveals the molecular architecture of the human prefoldin network

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Fabian Frommelt  

LAB HEAD: Matthias Gstaiger

PROVIDER: PXD035032 | Pride | 2024-03-11

REPOSITORIES: Pride