Phospho-proteomic Profiling Dataset of Chemical Perturbations in Multiple Biological Backgrounds
Dataset with its publication pending
- Organism: Homo sapiens
- Instrument: Q Exactive HF
Data-Independent Acquisition, mass spectrometry, proteomics, phospho-signaling, phosphoproteomics
Lab head: Malvina Papanastasiou
Submitter: Karen Christianson
The Library of Integrated Network-based Cellular Signatures (LINCS) Program aims to create a network-based understanding of biology by cataloging changes in gene expression and other cellular processes that occur when cells are exposed to a variety of perturbing agents. The goal of this project is to test the hypothesis that modulation of phosphorylation-mediated signaling events in response to perturbations can establish new cellular states by altering their epigenetic landscape. We have performed mass spectrometry (MS)-based proteomic assays that target quantitative readouts of phospho-signaling and chromatin modifications in cellular models, following perturbation by compounds with varying mechanisms of action. We have employed 128 compounds and treated 5 cancer and 2 neuronal cell lines. The resulting data collected and tools created have been contributed to the LINCS program for the purpose of making connections among disparate perturbations through phosphoproteomics and chromatin modification signatures in concert with other available data types. The data is publicly available on PanoramaPublic and can be queried using Clue (Broad Institute). Analysis of our data will inform novel therapeutic opportunities and synergies, as dysregulation of phospho-signaling and epigenetic systems are two of the most common molecular etiologies identified in a growing number of diseases.
Detailed P100 protocols can be found in Abelin et al., 2016, MCP and online at https://panoramaweb.org/wiki/LINCS/Overview%20Information/page.view?name=sops. Briefly, cells were lysed after drug treatment with a urea-based buffer that includes phosphatase inhibitors. Proteins (300-500 μg, depending on the cell type) extracted from the cell lysates underwent trypsin digestion followed by desalting using reversed solid phase extraction. Peptides were mixed with a quality-control set of synthetic isotope-labeled peptide standards to monitor the performance of subsequent steps. Phosphopeptide enrichment occurred on an AssayMAP Bravo automated liquid handling platform (Agilent, Santa Clara, CA) using IMAC chemistry. Samples were processed in a 96-well plate format. Prior to mass spectrometry analysis, a second set of synthetic isotope-labelled internal standards for targeted phosphopeptides were spiked into samples to ensure accurate quantification. Data were acquired using a DIA method as described in Abelin et al., 2016, MCP and ion chromatograms were extracted using Skyline (MacLean et al., 2014).
Drug signatures were profiled in various cancer cell lines (A375, YAPC, A549, MCF7, and PC3) and neuronal cell models (Astrocytes & NPCs). Drugs were grouped into four tranches: epigenetically active, neuroactive, kinase inhibitors and cardiotoxic compounds. A detailed description of their mechanism of action can be found online at https://panoramaweb.org/wiki/LINCS/Overview%20Information/page.view?name=LINCS%20PCCSE%20Overview and https://clue.io/touchstone. Drug stocks were dissolved in DMSO for treatment in cell line media and cells were treated in 6 well plates for 3 hours. For cell harvesting, cells from 2-4 wells of a 6 well plate were scraped in lysis buffer and combined per sample. All drug perturbations occurred in triplicates.
Created on 2/7/20, 3:39 PM