Dataset Information

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry

ABSTRACT: Quantitative proteomics employing mass spectrometry has become an indispensable tool in basic and applied life science research. Methods based on data-dependent acquisition have proved extremely valuable for qualitative proteome analysis but historically have struggled to achieve reproducible quantitative data. Targeted proteomics, most commonly implemented as selected reaction monitoring, has emerged as a powerful alternative and succeeded in providing a data independent approach for reproducible quantitative proteomics data but is limited in the number of proteins quantified. SWATH-MS is a recently introduced implementation of the data-independent acquisition strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, specificity) achieved in targeted proteomics but on the scale of thousands of proteins. While previous SWATH-MS studies have shown high intra-lab reproducibility, this has not been evaluated on an inter-lab basis. In this multi-laboratory evaluation study using data from 11 sites worldwide, we have demonstrated that using SWATH-MS we can consistently detect and quantify more than 4,000 proteins from HEK293 cells and that the quantitative protein data generated across laboratories is reproducible. Using synthetic peptide dilution series, we have shown that the sensitivity, dynamic range and reproducibility established with SWATH-MS methods are also uniformly achieved across labs. This study demonstrates that SWATH-MS is a reproducible technique that can be confidently deployed for large-scale protein quantification in life science research

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Ben Collins

LAB HEAD: Ruedi Aebersold

PROVIDER: PXD004886 | Pride | 2020-02-19

REPOSITORIES: Pride

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Publications

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry.

Collins Ben C BC Hunter Christie L CL Liu Yansheng Y Schilling Birgit B Rosenberger George G Bader Samuel L SL Chan Daniel W DW Gibson Bradford W BW Gingras Anne-Claude AC Held Jason M JM Hirayama-Kurogi Mio M Hou Guixue G Krisp Christoph C Larsen Brett B Lin Liang L Liu Siqi S Molloy Mark P MP Moritz Robert L RL Ohtsuki Sumio S Schlapbach Ralph R Selevsek Nathalie N Thomas Stefani N SN Tzeng Shin-Cheng SC Zhang Hui H Aebersold Ruedi R

Nature communications 20170821 1

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While pre ...[more]

PMID: 28827567

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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

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry

Publications

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry.

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