An integrated workflow for charting the human interaction proteome: insights into the PP2A system

Abstract

Protein complexes represent major functional units for the execution of biological processes. Systematic affinity purification coupled with mass spectrometry (AP-MS) yielded a wealth of information on the compendium of protein complexes expressed in Saccharomyces cerevisiae. However, global AP-MS analysis of human protein complexes is hampered by the low throughput, sensitivity and data robustness of existing procedures, which limit its application for systems biology research. Here, we address these limitations by a novel integrated method, which we applied and benchmarked for the human protein phosphatase 2A system. We identified a total of 197 protein interactions with high reproducibility, showing the coexistence of distinct classes of phosphatase complexes that are linked to proteins implicated in mitosis, cell signalling, DNA damage control and more. These results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP-MS studies on human protein complexes.

Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein–Protein Interaction Data*

“…Large-scale protein interaction screens have revolutionized our understanding of the basic principles of how proteins are organized in large protein complex machineries (1,28,29). However, in many cases, translation of this information to novel functional paradigms has been challenging.…”

Structure-Function Analysis of Core STRIPAK Proteins

“…PP2A acquires substrate and subcellular localization specificity via association with various scaffolding and regulatory subunits to form a number of different holoenzymes, most of which are trimers. In previous studies using affinity purification coupled to mass spectrometry, a portion of PP2A was also found in a higher order complex that we termed STRIPAK (striatin interacting phosphatase and kinase) (3,4). In addition to the catalytic subunit PP2A cat , its scaffolding subunit PP2A A and members of the striatin family of regulatory subunits (5), the core STRIPAK complex contains the striatin interactor Mob3 (6), the uncharacterized protein STRIP1, members of the germinal center kinase III (GCKIII) group (STK24, STK25, and MST4; Ref.…”

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-Based Strategy for Proteome-Wide Thermodynamic Analysis of Protein-Ligand Binding Interactions

“…Currently, the most widely used methods for proteome-wide analyses of protein-ligand binding interactions are those that combine an affinity purification step with a mass spectrometry-based proteomics analysis. Such methods have provided a wealth of information about protein-protein interaction networks in different proteomes (1)(2)(3)(4), and they have helped identify the protein targets of small molecules (5-7). However, a significant drawback to their use is the need for specially designed ligands to facilitate the affinity purification.…”