Leveraging a Multi-Omics Strategy for Prioritizing Personalized Candidate Mutation-Driver Genes: A Proof-of-Concept Study

The expression of mutant forms of proteins (e.g., oncogenes and tumor suppressors) has implications in cancer biology and clinical practice. Initial efforts have been made to characterize the transcription of tumor-mutated alleles; however, few studies have been reported to link tumor-mutated alleles to proteomics. We aimed to characterize the transcriptional and translational patterns of tumor-mutated alleles. We performed whole-exome sequencing, RNA-seq, and proteome profiling in a hyper-mutated patient of hepatocellular carcinoma. Using the patient as a model, we show that only a small proportion of tumor-mutated alleles were expressed. In this case, 42% and 3.5% of the tumor-mutated alleles were identified to be transcribed and translated, respectively. Compared with genes with germline variations or without mutations, somatic mutations significantly reduced protein expression abundance. Using the transcriptional and translational patterns of tumor-mutated alleles, we classified the mutations into four types, and only one type may be associated with the liver cancer and lead to hepatocarcinogenesis in the patient. Our results demonstrate how tumor-mutated alleles are transcribed and translated, and how the expression enables the classification of somatic mutations that cause cancer. Leveraging multiple ‘omics’ datasets provides a new avenue for understanding patient-specific mutations that underlie carcinogenesis.


Western blot of MSH2
Frozen tissues were homogenized using the Tissue Homogenizer (Omni, American) in ice-cold RIPA lysis buffer (50mM Tris base, pH 7.4, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10nM PMSF) containing protease inhibitor cocktail tablets (Roche, Switzerland). Then, the tissues were lysed at 4℃ for 30min in lysis buffer and the lysates were clarified by centrifugation at 15,000rpm at 4℃for 30min. Protein concentration was determined using the 2D Quantification Kit (Amersham Biosciences, Sweden). The protein samples (50ug in each well) were separated by SDS-PAGE and transferred to PVDF membrane.
The membranes were blocked with 5% non-fat dry milk in TBS-T buffer (20mM Tris, pH 7.6, 100mM NaCl, 0.5% Tween-20) overnight at 4℃, followed by 3 hours of incubation with the primary antibody (Santa Cruz, American, 1:100 dilution) in TBS-T buffer containing 5% non-fat dry milk at room temperature. After washing three times with TBS-T buffer, the membranes were incubated with an HRP-conjugated goat anti-mouse IgG as the secondary antibody (MULTISciences, China, 1:5000 dilution) for 1 hour at room temperature. The membranes were then washed three times in TBS-T buffer and the reactions were visualized with the ECL detection system. All of the analyses were repeated at least three times.

Protein extraction and in-solution digestion
The liver tissues were resuspended in ice-cold lysis buffer (8 M urea, 20 mM Tris-HCl, pH 8.0, 1mM Na2VO3; 5mM NaF; 20mM DTT, 1% protease inhibitor cocktail). Acid cleaned glass beads were added and the tissue protein was extracted by collision for 30s with 70Hz energy. The protein extraction were moved to a new tube and further sonicated for 200 W × 1 s (working) × 2 s (resting) × 30 circles. The suspension was centrifuged at 12000 g for 15 min at 4 °C. Protein concentration was determined by a BCA assay.
For in-solution digestion, the tissue lysates were first reduced with 10mM DTT for 4h at 37 °C and alkylated with 40mM IAA in dark for 1 hour. Excess IAA was quenched by adding 20mM of DTT. The urea concentration in the sample solution was reduced to 1M with 50 mM NH4HCO3, and proteins were digested with trypsin (Promega, USA) overnight. The protein to enzyme ratio was 100:1 and protein digestion was stopped by adding formic acid at 0.1% final concentration.

Serial peptide prefractionation by IEF and high pH reversed-phase chromatography
Tryptic peptides were fractionated according to the manufacturer's protocol using ImmobilinTM DryStrip, pH 3-10, 13cm (GE Healthcare) on an OFFGEL 3100 system (Agilent Technologies, USA). Twelve fractions were collected from the fractionator and then every three continuous fractions were concatenated into one faction, which resulted into 4 fractions from the IEF separation.
Mobile phases A (2% acetonitrile in water (v/v), adjusted pH to 10.0 using NH3·H20) and B (98% acetonitrile in water (v/v), adjusted pH to 10.0 using NH3·H20) were used to develop a 40 min gradient. The flow rate was 0.7 mL/min and the column oven was set as 45°C. Eluent was collected every minute. Then the fractions were dried under vacuum (Thermo Savant). Before MS identification, the peptides were reconstituted in 0.1% (v/v) FA, 2% (v/v) acetonitrile in water, and pooled in a discontinuous mode into 24 (for the acid end IEF fraction) or 12 fractions (for the other three IEF fractions).

Mass spectrometric analysis of peptide mixture
Peptide mixture was measured on an Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with an Easy-nLC nanoflow LC system (Thermo Fisher Scientific). Peptides were separated on a C18 column (3 µm, 100 Å, 75 um ID ×10 cm). Mobile phase A consisted of 0.1% FA, 2% acetonitrile in water, and mobile phase B consisted of 0.1% FA, 98% acetonitrile in water. The solvent gradient was set as follows: 5%-8% B, 3 min; 8%-22% B, 30min; 22%-32% B, 5 min; 32%-90% B, 1 min; 90% B, 6 min. For the Q-Exactive part, the source was operated at 2.2 kV. For full MS survey scan, AGC target was 3e6, scan range was from m/z 300 to 1400 with the resolution of 70,000. The 75 most intense peaks with charge state 2 and above were selected for sequencing and fragmented in the ion trap by HCD with normalized collision energy of 27%.
Exclude isotope item was on and dynamic exclusion time was set as 18s.

Selective reaction monitoring (SRM) validation
SRM transitions were calculated using Skyline software (https://skyline.gs.washington.edu) from the peptide amino acid sequence. At least the most intensity 8 fragment ions/each peptide were selected to setup MRM transitions. The MRM validation experiment was performed on Eksigent nanoLC-Ultra® 2D System and QTRAP 6500 system (AB SCIEX). And the MRM peaks were extracted and analyzed with Skyline software. The peptide with at least 8 MRM peaks was considered to be confirmed by MRM experiment.
For the mass spectrometric analysis, about 2 microgram peptide mixture was separated on an Eksigent nanoLC-Ultra® 2D System with a cHiPLC®-nanoflex system (Eksigent, USA) in trap elute mode. In each injection, the sample was desalted on a 200 µm x 6 mm trap chip and then eluted onto a 200 µm x 150 mm column chip for MS analysis. The media for both the trap and column chips were ChromXP C18-CL (3µm, 120Å, Eksigent). Peptides were separated using a linear gradient formed by A (2% ACN, 0.1% FA) and B (98% ACN, 0.1% FA) from 7-35% of B over 75 minutes at a flow of 300nL/min.
The MS analysis was performed on a QTRAP® 6500 system (AB SCIEX). The optimal acquisition parameters were as follows: curtain gas (30), ionspray voltage (2300V), ion source gas (15), interface heater temperature (150℃), collision gas (High), declustering potential (80), entrance potential (10) and collision cell exit potential (15). The resolution parameters of the first and the third quadrupole were set as "unit". The target ions were transmitted with a narrow window (0.7 Da). The dwell time was 20 ms for every transition.

MS database searching
All raw files of mass spectra were converted into mzXML and MGF files using the msconvert module in the Trans-Proteomic Pipeline (TPP v4.5.2). The MS/MS peak lists were searched using the Mascot v2.3.2 local server against the database containing sequences of all human proteins from Refseq (71,448 proteins, release 64) and the mutated sequences constructed from WES and RNA-seq data. The target-decoy strategy was applied to maintain the FDR less than 1% at the peptide level. For the database searching using Mascot, the monoisotopic mass was used for both peptide and fragment ions, with fixed modification (Carbamidomethyl/carbamidomethylation, +57.0214 Da) on cysteine and variable modification (Oxidation, +15.9949 Da) on methionine. Tryptic cleavage after Lys or Arg was selected and up to 2 missed cleavage sites were allowed. The precursor and fragment ion mass tolerance 20 ppm and 0.05 Da.
A normalized label-free quantitation method based on the extracted ion chromatograms (XICs) was applied to all confidently identified peptides by the software of SILVER, and then the cross-search between the cancer and normal samples were performed to avoid the randomly missing in the identifications. We also used the measure of spectral count (SC) 1 , which are the total number of MS/MS spectra acquired for peptides from a given protein, to quantify protein abundance.