The Hidden Story of Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma—Association with Clinical Outcome and Tumor Phenotypes

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.


TMT 11-Plex Labeling
The peptide content in each sample was determined using the quantitative colorimetric peptide assay kit (Thermo Fisher Scientific) to ensure equal amounts of material in each TMT channel. TMT labeling was performed according to manufacturer's instructions. Samples were resuspended in 100 µL of 200 mM TEAB and individual TMT 11-plex reagents were dissolved in 41 µL of dried ACN. Peptides were labeled by mixing the peptide solution with TMT 11-plex reagents for 1 h at room temperature. Reaction was quenched by adding 1 µL of 5% hydroxylamine and incubation at room temperature for 15 min. Then, the labeled peptides were mixed in a single tube, the volume was reduced in a speed vac, and the peptides were cleaned up using a C-18 Sep-Pak cartridge (Waters, Chromatography Europe, Etten-Leur, The Netherlands). The eluted peptides were dried in a speed vac and finally resuspended in 20 mM ammonium formiate prior to high pH fractionation.

Off Line High pH Fractionation
Each batch of TMT-11 labelled peptide was fractionated using a Phenomenex Aeris Widepore XB-C8 (3.6 µm, 2.1 × 100 mm) column on an 1100 Series HPLC (Agilent) operating at 80 µL/min. The mobile phases were solvent A: 20 mM ammonium formate and solvent B: 80% ACN-20% water containing 20 mM ammonium formate. Both solvents were adjusted to pH 10 with ammonium hydroxide. Separation was performed using the following gradient: 0 min 5% B; 1 min 20% B; 60 min 40% B; 90 min 90% B; 120 min 90% B. The column was operated at RT and the detection wavelength was 214 nm. Ninety-eight fractions were collected at 1 min intervals and further concatenated to 24 or 25 fractions, which were dried in a SpeedVac (Eppendorf). nLC-MS/MS analysis was performed on an Ultimate 3000 HPLC coupled to a Q Exactive HF-X mass spectrometer (Thermo Scientific, San Jose, CA, USA). Peptides from each fraction (1 µg) were loaded onto a trap column (Acclaim1 PepMap 100 pre-column, 75 µm, 2 cm, C18, 3 mm, 100 Å, Thermo Scientific) and then separated on an analytical column (EASY-Spray column, 25 cm, 75 µm i.d., PepMap RSLC C18, 2 mm, 100Å, Thermo Scientific) using a 120 min ACN gradient with 0.1% formic acid at a flow rate of 300 nL/min and a column temperature of 45 °C. Q Exactive HF-X mass spectrometer was set using the TMT node, as follows: full MS scans at m/z 350-1400 with a resolution of 120,000 at m/z 200, a target AGC value of 3 × 10 6 and IT of 50 ms, DDA selection of the 20 most intense ions for fragmentation in HCD collision cell with an NCE of 34 and MS/MS spectra acquisition in the Orbitrap analyzer at a resolution of 45,000 (at m/z 200) with a maximum IT of 86 ms, fixed first mass of 110 m/z, isolation window of 0.7 Da and dynamic exclusion of 30 s.
Quality controls were introduced to evaluate and maintain the performance of the nLC-MS/MS systems. A protein digest from Hela cells (Pierce HeLa Protein Digest Standard, Thermo Fisher Scientific) mixed with a standard peptide mixture (Pierce Peptide Retention Time Calibration Mixture) was measured every 10 LC-MS/MS analysis. This allowed the monitoring of peak width, retention time, base peak intensity, number of MS/MS, PSMs, peptides and proteins identified, and MS/MS success rate, among other parameters.

Data Analysis
Data were processed with Proteome Discoverer 2.3 (Thermo Fisher Scientific) using the Sequest HT search engine. The search was performed against the Homo sapiens UniProt revised database (2018-10-01) and the B-raf V600E mutant protein sequence. Cysteine carbamidomethylation was set as a fixed modification, while methionine oxidation and TMT 6plex at peptide N-terminus and lysine were set as variable modifications: peptide mass tolerance for the precursor ions and MS/MS spectra were 10 ppm and 0.02 Da, respectively. A maximum of two missed cleavage sites was accepted and FDR were set at 0.01 for identification at peptide level. The PD software allowed the introduction of reporter ion interferences for each batch of TMT 11-plex reagents as isotope correction factors in the quantification method.
The search results were directly processed in Perseus software [1]. A filtering criterion was set to keep the identified proteins with the quantified values of all reporter ions (no missing value) in the final identification list. The protein intensities were log2-transformed and normalized by subtracting the median intensity in each sample. The relative abundance values were obtained by subtracting the intensity of the protein in the reference sample.
For statistical analysis, samples were separated in two groups according to the levels of mutated B-raf: Group 1 (V600E_H), with B-raf > 1.65, and Group 2 (V600E_H), B-raf ≤ 1.65. The cut-off value (1.65) was selected by an ROC curve, where the ability of mutated B-raf to discriminate between long and short survivals (considering a three years' survival) was analyzed ( Figure S1). To make the ROC curve, patients of less than 40 years at the age of diagnosis were excluded. Kaplan-Meier survival analysis with log-rank, Breslow and Tarone Ware testing was used for univariate analysis between Groups 1 and 2. P-value < 0.05 was considered statistically significant. These analyses were performed using SPSS 25 (SPSS Inc, Chicago, IL, USA) software.
Differentially expressed proteins between V600E_H and 2 V600E_L were determined by Student t-test (two-tails). In this case, proteins quantified in at least three samples in each group were considered for the analysis and p-values < 0.01 dictated significant protein changes. Principal component analysis (PCA) was performed in R [2,3] ('FactoMineR' package) to visualize the behavior of DEPs. These proteins were included in the bioinformatics analysis, including hierarchical clustering ('pheatmap' R package; distance: Euclidean; linkage: average) and heat map generation. For functional analysis of the DEPs, the Ingenuity IPA Core Analysis was performed (Ingenuity, Qiagen). Figure S1. ROC curve analysis for the discrimination analysis between patients with long (more than three years) or short (less than three years) survival, according to B-raf V600E relative abundances measured by mass spectrometry on melanoma tumors. The cut-off (relative abundance of B-raf V600E higher than 1.65) was determined considering 100% of sensitivity and specificity for long survival patients. Low-expressing B-raf V600E tumors.