Biology subjects

Ruedi Aebersold

Publications and source records attributed to Ruedi Aebersold.

5 recordsLinked to original sources

bioRxiv2016

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

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 if large sample cohorts are comparatively analyzed. 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 technique consisting of a data-independent acquisition and a targeted data analysis 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 and accurate technique that can be confidently deployed for large-scale protein quantification in life science research.

Biochemistry↗

bioRxiv2016

A high density map for navigating the human Polycomb complexome

Polycomb group (PcG) proteins are major determinants of gene silencing and epigenetic memory in higher eukaryotes. Here, we used a robust affinity purification mass spectrometry (AP-MS) approach to systematically map the human PcG protein interactome, uncovering an unprecedented breadth of PcG complexes. The obtained high density protein interaction data identified new modes of combinatorial PcG complex formation with proteins previously not associated with the PcG system, thus providing new insights into their molecular function and recruitment mechanisms to target genes. Importantly, we identified two human PR-DUB de-ubiquitination complexes, which comprise the O-linked N-acetylglucosamine transferase OGT1 and a number of transcription factors. By further mapping chromatin binding of PR-DUB components genome-wide, we conclude that the human PR-DUB and PRC1 complexes bind distinct sets of target genes and impact on different cellular processes in mammals.

Systems Biology↗

bioRxiv2016

System-wide quantitative proteomics of the metabolic syndrome in mice: genotypic and dietary effects

Advances in mass spectrometry have made the quantitative measurement of proteins across multiple samples a reality, allowing for the study of complex biological systems such as the metabolic syndrome. Although deregulation of lipid metabolism and increased hepatic storage of triacylglycerides are known to play a part in the onset of the metabolic syndrome, its molecular basis and dependency on dietary and genotypic factors are poorly characterized. Here, we used a rich experimental design with two different mouse strains, dietary and metabolic perturbations to generate a compendium of quantitative proteome data, using three mass spectrometry strategies. The data recapitulates known properties of the metabolic system and indicate differential molecular adaptation of the two mouse strains to perturbations, contributing to a better understanding of the metabolic syndrome. We show that high-quality, high-throughput proteomic datasets provide an unbiased broad overview of complex systems upon perturbation.\n\nHIGHLIGHTSO_LIRich experimental design with two mouse strains, dietary and metabolic perturbations\nC_LIO_LIThree mass spectrometry proteomic data collection strategies in one data compendium\nC_LIO_LISWATH-MS data with systematic perturbation provide a broad unbiased view of the system\nC_LIO_LIThe data reveals differential adaptation of molecular systems in two mouse strains\nC_LI

Systems Biology↗

bioRxiv2016

Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle

The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network.

Biochemistry↗

bioRxiv2016

Automated SWATH Data Analysis Using Targeted Extraction of Ion Chromatograms

Targeted mass spectrometry comprises a set of methods able to quantify protein analytes in complex mixtures with high accuracy and sensitivity. These methods, e.g., Selected Reaction Monitoring (SRM) and SWATH MS, use specific mass spectrometric coordinates (assays) for reproducible detection and quantification of proteins. In this protocol, we describe how to analyze in a targeted manner data from a SWATH MS experiment aimed at monitoring thousands of proteins reproducibly over many samples. We present a standard SWATH MS analysis workflow, including manual data analysis for quality control (based on Skyline) as well as automated data analysis with appropriate control of error rates (based on the OpenSWATH workflow). We also discuss considerations to ensure maximal coverage, reproducibility and quantitative accuracy.

Bioinformatics↗