Multi-omic dissection of oncogenically active epiproteomes identifies drivers of proliferative or invasive breast tumors
Here we present a functional multi-omics method, interaction-Correlated Multi-omic Aberration Patterning or iC-MAP, which dissects intra-tumor heterogeneity and identifies in situ in real time the oncogenic consequences of multi-omics aberrations that drive proliferative/invasive tumor in individuals with poor prognosis. First, given epigenetic aberrations resulting from complex interactions between genetic susceptibility and environmental influences are the primary driving force of tumorigenesis, we applied our chromatin activity-based chemoproteomics (ChaC) method to characterize the tumor-phenotypic epiproteomes (epigenetic regulatory proteomes) in breast cancer (BC) patient tissues. A biotinylated ChaC probe UNC0965 that specifically binds to the oncogenically active histone methyltransferase G9a enabled the sorting/enrichment of a G9a-interacting epiproteome representing the predominant BC subtype in a tissue, which is separated from other cell types, especially non-malignant cells where G9a is less enzymatically active. ChaC then identified UNC0965-captured G9a interactors that are mostly involved in the oncogenic pathways associated with tumor cell viability and invasion. Using BC patient transcriptomic/genomic data we retrospectively identified the G9a interactor-encoding genes that show individualized iC-MAP in BC-subtypic patients with incurable metastatic disease, revealing essential drivers of proliferative or invasive BC phenotypes. Our iC-MAP findings can not only act as new diagnostic/prognostic markers to identify patient subsets with metastatic disease but also create precision therapeutic strategies that can match proliferative or invasive potential of individual patients.
Sample Processing Protocol
UNC0965 pull-downs and ChaC sample processing. 1 mg nuclear proteins extracted from either cell lines or clinical tissues were incubated with 2 nmole UNC0965 pre-coupled to 50 μl neutravidin-agarose (Thermo-Fisher) overnight at 4OC, and washed 3 times with 1 ml lysis buffer to remove non-specific proteins. For on-beads sampling and processing, in addition to 3 washings with lysis buffer, a washing buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl) was used 5 times to remove residual detergents. On-beads tryptic digestion was performed with 125 μl buffer containing 2 M urea, 50 mM Tris-HCl pH8.0, 1 mM DTT, 500 ng trypsin (Promega) for 30 min at room temperature on a mixer (Eppendorf). The tryptic digests were eluted twice with a 100 μl elution buffer containing 2 M urea, 50 mM Tris-HCl pH8.0, 5 mM iodoacetamide. Combined eluates were acidified with trifluoroacetic acid at final concentration of 1% (TFA, mass spec grade, Thermo-Fisher) and desalted by C18 stage tip. LC-MS/MS analysis. Desalted peptide mixtures were dissolved in 30 μl 0.1% formic acid (Thermo-Fisher), of which 4 μl containing the peptides from 60-100 μg total protein was injected and analyzed by a ultra2D nanoLC system (Eksigent) coupled to a Velos LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) or an Easy nanoLC 1000 coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). In the nanoLC-Velos setup, peptides were first loaded on to a 2 mm × 0.5 mm reverse-phase (RP) C18 trap column (Eksigent) at a flow rate of 1 μl/min, then eluted, and fractionated on a 25 cm C18 RP column (25 cm × 75 μm ID, C18, 3 μm) with a gradient of 5-40% buffer B (ACN and 0.1% formic acid) at a constant flow rate of 250 nl/min over 180 min. In the Easy nanoLC- Q Exactive setup, peptides were loaded on to a 15 cm C18 RP column (15 cm × 75 μm ID, C18, 2 μm, Acclaim Pepmap RSLC, Thermo-Fisher) and eluted with a gradient of 2-30% buffer B at a constant flow rate of 300 nl/min for 70 min followed by 30% to 80% B in 5 min and 80% B for 10 min. The Velos LTQ Orbitrap was operated in the positive-ion mode with a data-dependent automatic switch between survey Full-MS scan (m/z 300-1800) (externally calibrated to a mass accuracy of <5 ppm and a resolution of 60,000 at m/z 400) and CID MS/MS acquisition of the top 15 most intense ions. The Q-Exactive was also operated in the positive-ion mode but using a data-dependent top 20 method. Survey scans were acquired at a resolution of 70,000 at m/z 200. Up to the top 20 most abundant isotope patterns with charge ≥ 2 from the survey scan were selected with an isolation window of 2.0 m/z and fragmented by HCD with normalized collision energies of 27. The maximum ion injection time for the survey scan and the MS/MS scans was 250 ms and 120 ms, respectively and the ion target values were set to 1e6 and 2e5, respectively. Selected sequenced ions were dynamically excluded for 20 seconds.
Data Processing Protocol
Mass spec data and LFQ analysis. Mass spectral processing and peptide identification were performed on the Andromeda search engine in MaxQuant software (Version 22.214.171.124) against a human UniProt database. All searches were conducted with a defined modification of cysteine carbamidomethylation, with methionine oxidation and protein amino-terminal acetylation as dynamic modifications. Peptides were confidently identified using a target-decoy approach with a peptide false discovery rate (FDR) of 1% and a protein FDR of 5%. A minimum peptide length of 7 amino acids was required, maximally two missed cleavages were allowed, initial mass deviation for precursor ion was up to 7 ppm and the maximum allowed mass deviation for fragment ions was 0.5 Da. LFQ-based LC-MS/MS experiments were conducted in multiple replicates (2-3 biological replicates each with three technical replicates) on two sets of seven BC cell line of distinct PAM50-subtypes (luminal or BLBC/basal), or two tissue samples of the corresponding BC PAM50 subtypes paired with their adjacent normal (non-malignant) tissues. For LFQ analysis, a match between runs option was enabled and time window at 0.7 minutes. Data processing and statistical analysis were performed on Perseus (Version 126.96.36.199). Protein quantitation was performed on biological replicate runs and a two sample t-test statistics was used with a p-value of 5% to report statistically significant expression fold-changes.
Wrobel JA, Xie L, Wang L, Liu C, Rashid N, Gallagher KK, Xiong Y, Konze KD, Jin J, Gatza ML, Chen X. Multi-omic Dissection of Oncogenically Active Epiproteomes Identifies Drivers of Proliferative and Invasive Breast Tumors. iScience. 2019 17:359-378 PubMed: 31336272