Aaron J. Storey

Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver, and lung tissue.

Synthesis of NBI-961. Reagents and Conditions: a) NaOMe, MeOH 0 °C – rt. b) PPh3, DIAD, DCM 0 °C - rt. c) LDA, dry THF, -78 °C – rt. d) Na2CO3, Pd(dppf)Cl2.DCM, 4:1 THF:H2O. e) 7M NH3 in MeOH, NH4OH, 78 °C. f) K2CO3, Pd(dppf)Cl2.DCM, DMF, 80 °C.

KEA3 analysis that used the Cheng kinase-substrate interaction network in SUDHL5 and RIVA treated with NBI-961 relative to DMSO vehicle control using the top 25 proteins from each with a signficant loss of phosphorylation (FDR < 0.05; log2 fold change less than or equal to -1).

Differentially expressed proteins identified from the phospho-proteomic analysis in SUDHL5 treated with NBI-961 relative to DMSO vehicle control.

Raw data for kinase binding affinity assay with NBI-961.

1H NMR spectra of NBI-961. Purity of NBI-961 was determined by 1H NMR after dissolved in methanol. Spectra was recorded using an Agilent HPLC with a run time of 15.01 mins and a flow rate of 0.8 mL/min.

NBI-961 causes loss of NEK2 and NEK2 phosphorylation. A, B, Western blot analysis of NEK2 and phosphorylated NEK2 (p-NEK2) protein expression in NBI-961 treated RIVA (A) and U2932 (B) cell lines after 24 h. Quantification of band intensity normalized to ACTIN as mean ± SEM from three independent experiments shown as a bar graph below each blot. Adjusted P-values as determined by a one-way ANOVA with Dunnett’s multiple test correction for comparisons to DMSO vehicle treated control (VTC). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Short Tandem Repeat (STR) analysis of the DLBCL and benign B cell lines.

Differentially expressed proteins identified from the phospho-proteomic analysis in RIVA treated with NBI-961 relative to DMSO vehicle control.