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A Global Screen for Assembly State Changes of the Mitotic Proteome by SEC-SWATH-MS.

ABSTRACT: Living systems integrate biochemical reactions that determine the functional state of each cell. Reactions are primarily mediated by proteins. In proteomic studies, these have been treated as independent entities, disregarding their higher-level organization into complexes that affects their activity and/or function and is thus of great interest for biological research. Here, we describe the implementation of an integrated technique to quantify cell-state-specific changes in the physical arrangement of protein complexes concurrently for thousands of proteins and hundreds of complexes. Applying this technique to a comparison of human cells in interphase and mitosis, we provide a systematic overview of mitotic proteome reorganization. The results recall key hallmarks of mitotic complex remodeling and suggest a model of nuclear pore complex disassembly, which we validate by orthogonal methods. To support the interpretation of quantitative SEC-SWATH-MS datasets, we extend the software CCprofiler and provide an interactive exploration tool, SECexplorer-cc.

SUBMITTER: Heusel M

PROVIDER: S-EPMC7042714 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:It has become increasingly clear that biological functions depend not only on the identity and quantity, but also the correct interplay among the biomolecules that constitute the cell. Importantly, proteins as the key effectors in the cell dynamically organize into complexes. This study describes a workflow to interrogate the role of dynamic proteome organization in biological functions. Specifically, we here characterize the proteome of the HeLa CCL2 cell line in interphase and mitosis, including the measurement of the protein complex assembly state with the goal to screen for functional proteome rearrangement. We employ an optimized SEC-SWATH-MS workflow to profile protein elution along size exclusion chromatographic fractionations of mild proteome extracts. Largely maintaining the integrity of protein association into complexes, the method captures the proteomes contextual state via the measure of protein appearance at distinct molecular weight. Quantitatively accurate readout of polypeptide elution profiles is achieved by bottom-up DIA/SWATH mass spectrometry, facilitating sensitive detection of quantitatively context-re-wiring proteins. The study quantitatively profiles 5044 proteins across 390 SWATH-MS maps. Statistical analysis pinpoints several hundred proteins with significantly altered cellular context indicated by altered quantitative elution behavior across distinct apparent molecular weight ranges measured in SEC. The results are validated by the reconciliation of established cell biology such as CDK1 recruitment by cyclin B1 and the mitotic disassembly of nuclear pore complexes as presented in the accompanying manuscript. The high quality chromatographic data further delineate distinct cellular assemblies such as a novel mitotic intermediate of nuclear pore complex disassembly that was validated by orthogonal methods. The study sheds light on altered proteome state in the pivotal process of cell division at systems scale. In contrast to typical quantitative approaches to study the proteome, SEC-SWATH-MS captures the biophysical context state of proteins that is often associated with protein function. Thus, the data may well support the discovery and validation of novel aspects and effectors of modular proteome function along cell cycle progression. Our workflow and study build the capacity to investigate biological systems including their higher order organization on the level of proteins and in a systems-wide fashion which will help to elucidate how biochemical functions, responses and phenotypes are generated within a cell.

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A Global Screen for Assembly State Changes of the Mitotic Proteome by SEC-SWATH-MS.

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