Chemoproteomic Analysis of an NSD2-PWWP1 Chemical Probe

Dataset details

Project Title: Chemoproteomic Analysis of an NSD2-PWWP1 Chemical Probe

Keywords: chemoproteomics, epigenetics, chemical probe, histone methyltransferase

Project description: 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.

Methods:

Chemical Proteomics 

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.

Label-free quantitative mass spectrometry data analysis 

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. 

Brief Description of Data Files:

• .raw & .index files - raw proteomic data files - note: dataset has also been uploaded to ProteomeXchange Consortium via the PRIDE17 partner repository with the dataset identifier PXD017641. 
• mqpar.xml - provides parameters used for quantification with Maxquant, which can be found in the combined folder. 
• UP000005640_9606.fasta - UniProt reference used for Mazquant quantification of peptides
• NSD2_Chemoproteomics.Rproj & DEP_Analysis.R - R project & script file for differential analysis of Maxquant output with DEP. 
• methods.docx - additional details covering experimental method