Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition
TL;DR: It is shown that AP-SWATH is a robust label-free approach to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4 and proposed a scalable pipeline for systems biology studies.
Abstract: Changes to protein interactomes as a result of mutations to the bait protein or addition of a pharmacological inhibitor are robustly monitored with affinity purification coupled with data-independent acquisition–based mass spectrometry and an automated data analysis pipeline. Also in this issue, Collins et al. describe a similar method.
Citations
More filters
TL;DR: This tutorial provides guidelines on how to set up and plan a SWATH‐MS experiment, how to perform the mass spectrometric measurement and how to analyse SWath‐MS data using peptide‐centric scoring.
Abstract: Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH-MS is a specific variant of data-independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH-MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH-MS data, a strategy based on peptide-centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH-MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH-MS data using peptide-centric scoring. Furthermore, concepts on how to improve SWATH-MS data acquisition, potential trade-offs of parameter settings and alternative data analysis strategies are discussed.
613 citations
Cites background from "Mapping differential interactomes b..."
...…studies (Liu et al, 2015; Okada et al, 2016; Williams et al, 2016), clinical drug/perturbation studies (preprint: Litichevskiy et al, 2018; Tan et al, 2017; Keam et al, 2018) or exploratory basic research (Collins et al, 2013; Lambert et al, 2013; Parker et al, 2015a; Schubert et al, 2015b)....
[...]
TL;DR: DIA-Umpire enables targeted extraction of quantitative information based on peptides initially identified in only a subset of the samples, resulting in more consistent quantification across multiple samples.
Abstract: As a result of recent improvements in mass spectrometry (MS), there is increased interest in data-independent acquisition (DIA) strategies in which all peptides are systematically fragmented using wide mass-isolation windows ('multiplex fragmentation'). DIA-Umpire (http://diaumpire.sourceforge.net/), a comprehensive computational workflow and open-source software for DIA data, detects precursor and fragment chromatographic features and assembles them into pseudo-tandem MS spectra. These spectra can be identified with conventional database-searching and protein-inference tools, allowing sensitive, untargeted analysis of DIA data without the need for a spectral library. Quantification is done with both precursor- and fragment-ion intensities. Furthermore, DIA-Umpire enables targeted extraction of quantitative information based on peptides initially identified in only a subset of the samples, resulting in more consistent quantification across multiple samples. We demonstrated the performance of the method with control samples of varying complexity and publicly available glycoproteomics and affinity purification-MS data.
536 citations
TL;DR: Advances in mass spectrometry-based proteomics now enabling the measurement of multiple properties for thousands of proteins, including their abundance, isoform expression, turnover rate, subcellular localization, post-translational modifications and interactions are transforming the understanding of various cellular and physiological processes.
Abstract: The proteome is a dynamic system in which each protein has interconnected properties - dimensions - that together contribute to the phenotype of a cell. Measuring these properties has proved challenging owing to their diversity and dynamic nature. Advances in mass spectrometry-based proteomics now enable the measurement of multiple properties for thousands of proteins, including their abundance, isoform expression, turnover rate, subcellular localization, post-translational modifications and interactions. Complementing these experimental developments are new data analysis, integration and visualization tools as well as data-sharing resources. Together, these advances in the multidimensional analysis of the proteome are transforming our understanding of various cellular and physiological processes.
380 citations
TL;DR: In this paper, the authors present a compendium of highly specific assays covering more than 10,000 human proteins and enabling their targeted analysis in SWATH-MS datasets acquired from research or clinical specimens.
Abstract: Mass spectrometry is the method of choice for deep and reliable exploration of the (human) proteome. Targeted mass spectrometry reliably detects and quantifies pre-determined sets of proteins in a complex biological matrix and is used in studies that rely on the quantitatively accurate and reproducible measurement of proteins across multiple samples. It requires the one-time, a priori generation of a specific measurement assay for each targeted protein. SWATH-MS is a mass spectrometric method that combines data-independent acquisition (DIA) and targeted data analysis and vastly extends the throughput of proteins that can be targeted in a sample compared to selected reaction monitoring (SRM). Here we present a compendium of highly specific assays covering more than 10,000 human proteins and enabling their targeted analysis in SWATH-MS datasets acquired from research or clinical specimens. This resource supports the confident detection and quantification of 50.9% of all human proteins annotated by UniProtKB/Swiss-Prot and is therefore expected to find wide application in basic and clinical research. Data are available via ProteomeXchange (PXD000953-954) and SWATHAtlas (SAL00016-35).
378 citations
ETH Zurich1, Buck Institute for Research on Aging2, University of Zurich3, Institute for Systems Biology4, Johns Hopkins University School of Medicine5, University of California, San Francisco6, Lunenfeld-Tanenbaum Research Institute7, University of Toronto8, Washington University in St. Louis9, Kumamoto University10, Macquarie University11
TL;DR: This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.
Abstract: 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 previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification. SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories.
372 citations
References
More filters
TL;DR: Three methods of performing normalization at the probe intensity level are presented: a one number scaling based algorithm and a method that uses a non-linear normalizing relation by comparing the variability and bias of an expression measure and the simplest and quickest complete data method is found to perform favorably.
Abstract: Motivation: When running experiments that involve multiple high density oligonucleotide arrays, it is important to remove sources of variation between arrays of non-biological origin. Normalization is a process for reducing this variation. It is common to see non-linear relations between arrays and the standard normalization provided by Affymetrix does not perform well in these situations. Results: We present three methods of performing normalization at the probe intensity level. These methods are called complete data methods because they make use of data from all arrays in an experiment to form the normalizing relation. These algorithms are compared to two methods that make use of a baseline array: a one number scaling based algorithm and a method that uses a non-linear normalizing relation by comparing the variability and bias of an expression measure. Two publicly available datasets are used to carry out the comparisons. The simplest and quickest complete data method is found to perform favorably. Availabilty: Software implementing all three of the complete data normalization methods is available as part of the R package Affy, which is a part of the Bioconductor project http://www.bioconductor.org. Contact: bolstad@stat.berkeley.edu Supplementary information: Additional figures may be found at http://www.stat.berkeley.edu/∼bolstad/normalize/ index.html
8,324 citations
TL;DR: P16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein, and inhibits the catalytic activity of theCDK4/cyclin D enzymes.
Abstract: The division cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). The sequential activation of individual members of this family and their consequent phosphorylation of critical substrates promotes orderly progression through the cell cycle. The complexes formed by CDK4 and the D-type cyclins have been strongly implicated in the control of cell proliferation during the G1 phase. CDK4 exists, in part, as a multi-protein complex with a D-type cyclin, proliferating cell nuclear antigen and a protein, p21 (refs 7-9). CDK4 associates separately with a protein of M(r) 16K, particularly in cells lacking a functional retinoblastoma protein. Here we report the isolation of a human p16 complementary DNA and demonstrate that p16 binds to CDK4 and inhibits the catalytic activity of the CDK4/cyclin D enzymes. p16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein.
3,716 citations
TL;DR: In this paper, a small-molecule bromodomain inhibitor, JQ1, was used to identify BET proteins as regulatory factors for c-Myc oncoprotein.
2,500 citations
TL;DR: A new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest is presented, using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data-independent acquisition method.
2,358 citations
TL;DR: The recent determination of the structure of eIF4E at atomic resolution has provided insight about how translation is initiated and regulated and suggests that eif4F is also implicated in malignancy and apoptosis.
Abstract: ▪ Abstract Eukaryotic translation initiation factor 4F (eIF4F) is a protein complex that mediates recruitment of ribosomes to mRNA. This event is the rate-limiting step for translation under most circumstances and a primary target for translational control. Functions of the constituent proteins of eIF4F include recognition of the mRNA 5′ cap structure (eIF4E), delivery of an RNA helicase to the 5′ region (eIF4A), bridging of the mRNA and the ribosome (eIF4G), and circularization of the mRNA via interaction with poly(A)-binding protein (eIF4G). eIF4 activity is regulated by transcription, phosphorylation, inhibitory proteins, and proteolytic cleavage. Extracellular stimuli evoke changes in phosphorylation that influence eIF4F activity, especially through the phosphoinositide 3-kinase (PI3K) and Ras signaling pathways. Viral infection and cellular stresses also affect eIF4F function. The recent determination of the structure of eIF4E at atomic resolution has provided insight about how translation is initiat...
2,122 citations