Chemoproteomic Analysis of an NSD2-PWWP1 Chemical Probe

<p><strong>Dataset details</strong></p> <p><strong>Project Title: </strong>Chemoproteomic Analysis of an NSD2-PWWP1 Chemical Probe</p> <p><strong>Keywords: </strong>chemoproteomics, epigenetics, chemical probe, histone methyltransferase</p> <p><strong>Project description:&nbsp;</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.</p> <p><strong>Methods:</strong></p> <p><strong>Chemical Proteomics</strong>&nbsp;</p> <p>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&nbsp;KCl, 1% Triton X-100 + a protease inhibitor cocktail containing aprotinin, leupeptin,&nbsp;pepstatin&nbsp;A, and E-64) and passed through a&nbsp;25 gauge&nbsp;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&deg;C. Cleared supernatant was diluted to 150 mM&nbsp;KCl&nbsp;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 &micro;M UNC7145, or 20 &micro;M UNC6934 (final concentration) for 1 hour with rotation at 4&deg;C. For each sample, 25 &micro;l of M270&nbsp;Dynabeads&nbsp;(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 &micro;M UNC7096 (biotinylated probe) for 1 hour at 4 &deg;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&deg;C with 2 &micro;g of mass spectrometry grade trypsin (Promega). The following morning an additional 2 &micro;g of trypsin was added to each sample and incubated at 37&deg;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&nbsp;Thermo Scientific LTQ Orbitrap Velos.</p> <p><strong>Label-free quantitative mass spectrometry data analysis&nbsp;</strong></p> <p>Raw MS/MS files were searched and quantified using&nbsp;Maxquant&nbsp;version 1.6.7.0 using the UP000005640&nbsp;Uniprot&nbsp;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&nbsp;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.&nbsp;</p> <p>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.&nbsp;</p> <p><strong>Brief Description of Data Files:</strong></p> <ul> <li><em>.raw &amp; .index files</em> - raw proteomic data files - note: dataset has also been uploaded to ProteomeXchange&nbsp;Consortium via the&nbsp;PRIDE17&nbsp;partner repository with the dataset identifier PXD017641.&nbsp;</li> <li><em>mqpar.xml </em>- provides parameters used for quantification with Maxquant, which can be found in the combined folder.&nbsp;</li> <li><em>UP000005640_9606.fasta</em> - UniProt reference used for Mazquant quantification of peptides</li> <li><em>NSD2_Chemoproteomics.Rproj &amp; DEP_Analysis.R</em> - R project &amp; script file for differential analysis of Maxquant output with DEP.&nbsp;</li> <li><em>methods.docx</em> - additional details covering experimental method</li> </ul> <p>&nbsp;</p>