Global proteome rearrangement screen in interphasic and mitotic HeLa CCL2 cell line by SEC-SWATH-MS
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ABSTRACT: 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.
INSTRUMENT(S): TripleTOF 6600
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture, Cervix Epithelium
DISEASE(S): Cervix Carcinoma
SUBMITTER: 
Moritz Heusel
LAB HEAD: Prof. Ruedi Aebersold
PROVIDER: PXD010288 | Pride | 2019-05-10
REPOSITORIES: Pride
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