DOI: 10.5281/zenodo.5153405
title: Chemoproteomic Analysis of an NSD2-PWWP1 Chemical Probe

description: <strong>Dataset details</strong> <strong>Project Title: </strong>Chemoproteomic Analysis of an NSD2-PWWP1 Chemical Probe <strong>Keywords: </strong>chemoproteomics, epigenetics, chemical probe, histone methyltransferase <strong>Project description: </strong>Here we use competitive chemoproteomics pulldowns followed by label-free quantitative LC-MS/MS to assess target engagement and selectivity profiles of UNC6934 and UNC7145, a chemical probe targeting the PWWP1 domain of NSD2 and its negative control counterpart, respectively. To this end, we used a biotinylated probe derivative (UNC7096) for streptavidin pulldowns from KMS-11 multiple myeloma cell lysates, including in the context of UNC7145 or UNC6934 competition. <strong>Methods:</strong> <strong>Chemical Proteomics</strong> To prepare whole cell lysates, KMS11 cells were washed 2 times with 1x PBS, lysed by resuspension in high-salt lysis buffer (20 mM HEPES pH 7.5, 350 mM KCl, 1% Triton X-100 + a protease inhibitor cocktail containing aprotinin, leupeptin, pepstatin A, and E-64) and passed through a 25 gauge needle 5 times followed by a 20 min incubation on ice. Cell lysates were cleared by centrifugation at 18 000 x g for 20 minutes at 4°C. Cleared supernatant was diluted to 150 mM KCl and 0.4% Triton X-100 with 20mM HEPES pH7.5 including fresh protease inhibitors. Sample protein concentrations were determined using the BCA assay (ThermoScientific). For each pulldown, 3 mg of cell lysate was pre-incubated with either DMSO control, 20 µM UNC7145, or 20 µM UNC6934 (final concentration) for 1 hour with rotation at 4°C. For each sample, 25 µl of M270 Dynabeads (ThermoScientific) were prepared by washing three times in low salt wash buffer (10 mM Tris-HCl pH7.9, 100 mM NaCl, 0.1% NP-40), followed by incubation with 1 µM UNC7096 (biotinylated probe) for 1 hour at 4 °C. The unbound biotinylated compound was removed by 3 washes with low salt buffer. UNC7096 bound beads were then added to each sample followed by incubation 1 hour with rotation at 4oC. Beads were then washed 3 times with low-salt wash buffer followed by 2 washes with 50mM ammonium bicarbonate. On-bead digestion was performed by overnight incubation at 37°C with 2 µg of mass spectrometry grade trypsin (Promega). The following morning an additional 2 µg of trypsin was added to each sample and incubated at 37°C for 4-6 hours. The supernatant, containing digested peptides, was collected. Beads were then washed twice with water and supernatant pooled with digested peptides. Samples were then acidified with formic acid to a final concentration of 2% final concentration and flash frozen prior drying under vacuum before being run on a Thermo Scientific LTQ Orbitrap Velos. <strong>Label-free quantitative mass spectrometry data analysis </strong> Raw MS/MS files were searched and quantified using Maxquant version 1.6.7.0 using the UP000005640 Uniprot human database (containing 20,605 protein entries, last modified November 5, 2019) with label-free quantification enabled and variable modifications oxidized methionine (+15.9949 Da) and deamidated asparagine (+0.9840) set. First search peptide tolerance and main search peptide tolerance were set at 30 and 6 ppm, respectively. For all other parameters default settings were used. Differential enrichment analysis was performed using the DEP package (v1.8.0) in R (v3.5.1). Briefly, samples were filtered for proteins identified in 2 out of 3 replicates of at least one condition, normalized by variance stabilizing normalization and tested for differential enrichment relative to pulldowns competed with DMSO vehicle control. <strong>Brief Description of Data Files:</strong> <em>.raw & .index files</em> - raw proteomic data files - note: dataset has also been uploaded to ProteomeXchange Consortium via the PRIDE17 partner repository with the dataset identifier PXD017641. <em>mqpar.xml </em>- provides parameters used for quantification with Maxquant, which can be found in the combined folder. <em>UP000005640_9606.fasta</em> - UniProt reference used for Mazquant quantification of peptides <em>NSD2_Chemoproteomics.Rproj & DEP_Analysis.R</em> - R project & script file for differential analysis of Maxquant output with DEP. <em>methods.docx</em> - additional details covering experimental method
description: Acknowledgements The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from; AbbVie, Bayer AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Merck & Co (aka MSD outside Canada and US), Pfizer, Takeda and Wellcome [106169/ZZ14/Z]. We acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for a postdoctoral fellowship awarded to DD. This work was supported by the National Cancer Institute, NIH (grant R01CA242305) to L.I.J. MS gratefully acknowledge NSERC (grant RGPIN-2019-04416). Research in EpiCypher was supported by NIH grants R44GM117683 and R44GM116584. This work was supported by Cancer Research Society operating grant (25418) to D.B-L. We thank Taraneh Hajian for purfying proteins and Levon Halabelian for providing fluorescein-labelled dsDNA. We thank the University of North Carolina's Department of Chemistry Mass Spectrometry Core Laboratory, especially Diane Wallace, for their assistance with mass spectrometry analysis. The Mass Spectrometry Core Laboratory is supported by the National Science Foundation under Grant No. (CHE-1726291). Research reported in this publication was supported in part with funding by the University of North Carolina's School of Medicine Office of Research. We thank Frances Potjewyd for reviewing the primary synthesis data supporting this manuscript. Receptors, channels and transporters binding profiles were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2018-00023-C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L. Roth at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. For experimental details please refer to the PDSP web site https://pdsp.unc.edu/ims/investigator/web/.