D IAGNOSTICO DE LAS ACTIVIDADES REALIZADAS DENTRO DEL PROCESO Y EL APORTE

2.2.4.1 Western blot

Cells were harvested in RIPA buffer (Pierce) and the lysate sonicated using a Bioruptor Plus (Diagenode) for 2 minutes (30 seconds on/30 seconds off) to degrade the DNA. Protein was then quantified using a Direct Detect Spectrometer (Millipore). Samples were incubated at 70°C for 10 minutes in the presence of 1X LDS sample buffer (Invitrogen) and 1X NuPage sample reducing agent (Invitrogen) and loaded on NuPAGE 4-12% bis- Tris gels (Invitrogen). A Precision Plus Protein Standards molecular weight marker (BioRad) was used for determination of protein sizes. The gel tank apparatus was filled with 1X MOPS-SDS (3-(N-morpholino)propanesulfonic acid-sodium dodecyl suphate) running buffer (Invitrogen) and a voltage of 120 V applied for varying durations depending on the target protein size. Proteins were then transferred to nitrocellulose membranes using the iBlot® 2 Dry Transfer System (Invitrogen), or an overnight wet transfer (for TET2). The membrane was blocked using Odyssey Blocking Buffer (Li-Cor) for 1 hour at room temperature, and incubated with primary antibody overnight at 4°C. All primary antibodies (listed in Table 2.1) were used at a concentration of 1:1000, aside from the ERα antibody which was used at a concentration of 1:100. The membrane was then washed in Tris-buffered saline (50 mM Tris, 150 mM NaCl) containing 0.1% Tween-20 (TBS-T), incubated with fluorescent secondary antibodies (IRDye® 800 CW Goat anti- Mouse IgG 1:5,000 or IRDye® 680LT Goat anti-Rabbit 1:20,000, both Li-Cor) for 45 minutes at room temperature, and washed once more in TBS-T before imaging using the Odyssey CLx Imaging System (Li-Cor). Images were taken with the automated capture option of the Image Studio Version 4.0 software.

2.2.4.2 Parallel Reaction Monitoring (PRM)

2.2.4.2.1 Sample preparation and mass spectrometry

PRM sample preparation, method development and mass spectrometry (MS) analysis were performed by Dr Carmen Gonzalez Tejedo (Proteomics Core Facility, CRUK-CI).

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Surrogate peptides unique to the target proteins of interest (ER, TET2, GATA3 and Actin) were chosen and stable-isotope-labelled versions of these peptides were synthesised as SpikeTides™ peptides by JPT Peptide Technologies, GmbH (Berlin, Germany). Cells were washed twice in cold PBS and harvested in PBS containing protease inhibitors (Roche). Cells were lysed and peptides digested with trypsin, and a mix of stable isotope- labelled peptide standards was added to the mixture. Mixtures were desalted using either Ultra-Micro C18 Spin Columns (Harvard Apparatus) or cartridges from an iST Sample Preparation Kit (Preomics) and reconstituted in either 3% acetonitrile/0.1% formic acid or the iST Sample Preparation Kit load buffer (Preomics). A Pierce Peptide Retention Time Calibration Mixture containing 15 synthetic heavy peptides mixed at an equimolar ratio (Thermo Scientific) was added to each sample at a final concentration of 20 fmol of peptides per 2 µg of total protein to assess chromatography performance and optimise scheduled MS acquisition windows. Diluted peptide mixtures were analysed by liquid chromatography mass spectrometry (LC-MS) on a Dionex Ultimate 3000 UHPLC system coupled to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). Scheduled PRM transitions used a retention time window of 120 seconds. All samples were analysed in triplicate in the mass spectrometer.

2.2.4.2.2 PRM data processing and bioinformatic analysis

Data processing and bioinformatic analysis of PRM data was performed by Dr Carmen Gonzalez Tejedo (Proteomics Core Facility, CRUK-CI). All raw files were processed using Skyline-daily software v.19.0.9.190 (MacCoss Lab, University of Washington) for the generation of extracted-ion chromatograms and peak integration. Peak integrations were reviewed manually and transitions from analyte peptides were confirmed by the same retention times of the endogenous peptides and heavy stable isotope-labelled peptides time in a pre-selected retention time window. At least three transition ion peak area intensities were integrated and summed for each peptide (heavy and endogenous). The ratio of endogenous/heavy peak areas was calculated and the average of three independent injections of every sample was calculated to obtain a final quantification value for each peptide. Data were exported from Skyline for analysis and plotting using an in- house R script to calculate fold changes and p-values between different experimental conditions. Quantitative values obtained for actin peptides were used to normalise the data between different conditions.

41 2.2.4.3 Full proteome analysis

2.2.4.3.1 Sample preparation and mass spectrometry

Full proteome analysis was carried out by the Proteomics Core Facility (CRUK-CI), as described in Papachristou et al. (2018). Briefly, cells were washed twice in cold PBS and harvested in PBS containing protease inhibitors (Roche). Cells were sonicated in 200 μl of 0.1 M tetraethylammonium bromide (TEAB), 0.1% SDS (sodium dodecyl suphate) buffer followed by probe sonication and boiling at 95 °C. Protein concentration was estimated using a Bradford assay (BIO-RAD-Quick start) according to manufacturer’s instructions. For each sample, 90 μg of total protein was reduced for 1 hour at 60°C by the addition of 2 μl 50 mM tris-2-carboxyethyl phosphine (TCEP, Sigma). Cysteines were blocked for 10 minutes at room temperature with the addition of 1 μl 200 mM methyl methanethiosulfonate (MMTS, Sigma). Proteins were digested overnight at 37°C in trypsin (Pierce) solution, added at a ratio of ~30:1 protein:trypsin. The following day peptides were labelled using the TMT (tandem mass tag) 11plex reagents (Thermo Scientific) with a randomised design. Peptides were fractionated on a Dionex Ultimate 3000 system at high pH using the X-Bridge C18 column (Waters) with 1% gradient. UV signal was recorded at 280 and 215 nm and fractions were collected in a peak-dependent manner. Peptide fractions were analysed on a Dionex Ultimate 3000 UHPLC system coupled with a nano- ESI Fusion Lumos (Thermo Scientific).

2.2.4.3.2 Full proteome data processing and bioinformatic analysis

Data processing of full proteome results was carried out by the Proteomics Core Facility (CRUK-CI) according to Papachristou et al. (2018). Raw MS data was processed with the SequestHT search engine on the Proteome Discoverer 2.1 software for peptide and protein identifications. The node for SequestHT included the following parameters: Precursor Mass Tolerance 20 ppm, Fragment Mass Tolerance 0.5 Da. Dynamic Modifications were Oxidation of M (+15.995 Da), Deamidation of N, Q (+0.984 Da) and Static Modifications were TMT6plex at any N-Terminus, K (+229.163 Da), Methylthio at C (+45.988). The Reporter Ion Quantifier node included a TMT 6plex (Thermo Scientific Instruments) Quantification Method. Further bioinformatic analysis was carried out by Dr Kamal Kishore (Bioinformatics Core Facility, CRUK-CI). Pre-processed quantitative datasets (peptide or protein-level intensities) generated by Proteome Discoverer were imported into R and data analysed using the qPLEXanalyzer tool (Papachristou et al.

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2018), which uses analysis based on limma (an R/Bioconductor package) to identify differentially abundant proteins.