Building the Connectivity Map of epigenetics: Chromatin profiling by quantitative targeted mass spectrometry

Creech, Amanda L.; Taylor, Jordan E.; Maier, Verena; Wu, Xiaoyun; Feeney, Caitlin M.; Udeshi, Namrata D.; Peach, Sally E.; Boehm, Jesse S.; Lee, Jeannie T.; Carr, Steven A.; Jaffe, Jacob D.

doi:10.1016/j.ymeth.2014.10.033

Cited by 59 publications

(76 citation statements)

References 13 publications

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“…For example, it would be interesting to generate chromatin signature data on epigenetically active compounds in our global chromatin profiling assay (35,59) along with P100, thus providing measures of processes that are directly targeted by the molecules together with measures of secondary process that might also be of interest. Our demonstration of signaling responses for these compound classes (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…This assay is a phosphosignaling analog of our Global Chromatin Profiling assay (35,36) that has already proven to be of great utility. We believe that the P100 assay will prove highly useful for classification and stratification of drug and genetic mechanisms as facilitated through comparison of phosphoproteomic signatures of known chemical and genetic perturbations to those of novel perturbations or those where mechanistic insight is currently lacking.…”

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confidence: 99%

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Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes

Abelin

Patel

Lü

et al. 2016

Molecular & Cellular Proteomics

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110

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Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ϳ95% of the reduced-representation phosphopeptide probes to be detected in ϳ200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes

Abelin

Patel

Lü

et al. 2016

Molecular & Cellular Proteomics

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110

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“…Many diseases are known to cause changes in their dynamic PTM signaling profile, specifically in cancer affecting phosphorylation patterns which provides interesting experimental designs when using uDIA for their analysis [76,78,79]. Other studies have focused on histone modifications, such as in an elegant PRM study published by Jaffe and colleagues describing the targeted quantification histone PTMs [75], but also recent uDIA studies [80,81]. uDIA acquisitions for PTM work also enable strategies to determine PTM site occupancy as recently presented by Meyer et al [52].…”

Section: Application Of Dia/swath Workflows To Clinically Relevantmentioning

confidence: 99%

Clinical applications of quantitative proteomics using targeted and untargeted data-independent acquisition techniques

Meyer

Schilling

2017

Expert Review of Proteomics

132

105

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Introduction While selected/multiple-reaction monitoring (SRM or MRM) is considered the gold standard for quantitative protein measurement, emerging data-independent acquisition (DIA) using high-resolution scans have opened a new dimension of high-throughput, comprehensive quantitative proteomics. These newer methodologies are particularly well suited for discovery of biomarker candidates from human disease samples, and for investigating and understanding human disease pathways. Areas covered This article reviews the current state of targeted and untargeted DIA mass spectrometry-based proteomic workflows, including SRM, parallel-reaction monitoring (PRM) and untargeted DIA (e.g., SWATH). Corresponding bioinformatics strategies, as well as application in biological and clinical studies are presented. Expert commentary Nascent application of highly-multiplexed untargeted DIA, such as SWATH, for accurate protein quantification from clinically relevant and disease-related samples shows great potential to comprehensively investigate biomarker candidates and understand disease.

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“…The technique goes by various monikers such as PRM (parallel reaction monitoring), MRM-HR (multiple reaction monitoring, high resolution), HR-MRM, etc. Use of full scan MS/MS spectra in targeted assays allows for selection of many interference-free transitions for quantification and unambiguous identification of analytes, including localization of sites of post-translational modifications on peptides [5]. While perhaps not as sensitive as triple quadrupole instrumentation, high resolution instrumentation allows for more selectivity and less potential for interference.…”

Section: Introductionmentioning

confidence: 99%

Transitioning from Targeted to Comprehensive Mass Spectrometry Using Genetic Algorithms

Jaffe

Feeney

Patel

et al. 2016

J. Am. Soc. Mass Spectrom.

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Targeted proteomic assays are becoming increasingly popular due to their robust quantitative applications enabled by internal standardization, and they can be routinely executed on high performance mass spectrometry instrumentation. However, these assays are typically limited to 100s of analytes per experiment. Considerable time and effort are often expended in obtaining and preparing samples prior to targeted analyses. It would be highly desirable to detect and quantify 1000s of analytes in such samples using comprehensive mass spectrometry techniques (e.g., SWATH and DIA) while retaining a high degree of quantitative rigor for analytes with matched internal standards. Experimentally, it is facile to port a targeted assay to a comprehensive data acquisition technique. However, data analysis challenges arise from this strategy concerning agreement of results from the targeted and comprehensive approaches. Here, we present the use of genetic algorithms to overcome these challenges in order to configure hybrid targeted/comprehensive MS assays. The genetic algorithms are used to select precursor-to-fragment transitions that maximize the agreement in quantification between the targeted and the comprehensive methods. We find that the algorithm we used provided across-the-board improvement in the quantitative agreement between the targeted assay data and the hybrid comprehensive/targeted assay that we developed, as measured by parameters of linear models fitted to the results. We also found that the algorithm could perform at least as well as an independently-trained mass spectrometrist in accomplishing this task. We hope that this approach will be a useful tool in the development of quantitative approaches for comprehensive proteomics techniques.

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Building the Connectivity Map of epigenetics: Chromatin profiling by quantitative targeted mass spectrometry

Cited by 59 publications

References 13 publications

Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes

Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes

Clinical applications of quantitative proteomics using targeted and untargeted data-independent acquisition techniques

Transitioning from Targeted to Comprehensive Mass Spectrometry Using Genetic Algorithms

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