Dataset Information

SEC-SWATH-MS HEK293 - Complex-centric proteome profiling by SEC-SWATH-MS

ABSTRACT: Increasingly, biochemical co-fractionation-based approaches are used to study interactomes and protein complexes at high throughput. The devised methods facilitate the qualitative assignment and prediction of hundreds of putative cellular assemblies in one experiment and without dependency on genetic engineering to introduce affinity tags. The present dataset consists of a native proteome extracted by mild lysis from the HEK293 cell line and fractionated into 80 fractions along high resolution size exclusion chromatography, and each fraction analyzed via bottom-up SWATH mass spectrometry. In multi-tiered targeted analysis, from this fragment ion level chromatographic data, quantitative complex assembly information of the proteome is reconstructed in three steps, (i) peptide-centric detection of peptide analytes within each SEC fraction based on fragment ion co-elution groups; (ii) protein-centric detection of protein elution in SEC based on fragment peptide co-elution groups in SEC; (iii) complex-centric detection and quantification of protein complexes and -variants based on component subunit protein co-elution groups in SEC. The data delineate a global picture of quantitative complex formation within a human proteome, including deconvolution of novel subversions and assembly intermediates of critical cellular complexes with essential functions.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Moritz Heusel

LAB HEAD: Rudolf Aebersold

PROVIDER: PXD007038 | Pride | 2019-01-11

REPOSITORIES: Pride

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Publications

Complex-centric proteome profiling by SEC-SWATH-MS.

Heusel Moritz M Bludau Isabell I Rosenberger George G Hafen Robin R Frank Max M Banaei-Esfahani Amir A van Drogen Audrey A Collins Ben C BC Gstaiger Matthias M Aebersold Ruedi R

Molecular systems biology 20190114 1

Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consist ...[more]

PMID: 30642884

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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.

Dataset Information

SEC-SWATH-MS HEK293 - Complex-centric proteome profiling by SEC-SWATH-MS

Publications

Complex-centric proteome profiling by SEC-SWATH-MS.

Similar Datasets

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