Categorías de análisis del objetivo 1: identificación y análisis de los desarrollos temáticos

Proteomics studies in this thesis were performed by the analysis of isolated proteins under three different conditions; untreated, stimulated, and with the presence of the isolated compounds.

2.8.1 Two-dimensional gel electrophoresis

The protein samples were resuspended in sample buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 65 mM DTT, 1.5% v/v ampholytes, and a trace of bromophenol blue) and centrifuged at 13,000 rpm for 1 hour. The clear protein containing supernatant was collected and the undissolved pellet was discarded. Protein was quantified using 2D Quant Kit according to the manufacturer’s protocol.

To load the protein sample into the IPG strips, 125 µl of the sample (containing 150 µg protein) was pipetted along the channel of rehydration tray, followed by the application of the IPG (7 cm) strip onto the sample. The strips were incubated at room temperature for at least 16 hours to allow the complete rehydration of the strips. The proteins were subsequently separated according to their isoelectric points using the Protean IEF System (Bio-Rad Laboratories, Hertfordshire, UK). The isoelectric focusing (IEF) condition were 100 V for 40 min, 200 V for 20 min, 450 V for 15 min, 750 V for 15 min, reaching 2000 V in 10 min, for a total of 4000 Vh (max 0.125 mA and 0.125 W per strip). Following the IEF, the strips were equilibrated in DTT buffer (6 M urea, 50 mM Tris pH 6.8, 2% (w/v) SDS, 1% w/v DTT, 30% (v/v) glycerol, containing a trace of bromophenol blue) for 20 minutes. The strips were then transferred to a iodoacetamide buffer (6 M urea, 50 mM Tris pH 6.8, 2% w/v SDS, 2.5% w/v iodoacetamide, 30% v/v glycerol, and a trace of bromophenol blue) for further 20 minutes.

87 The second separation of the proteins was performed by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) using a 12% polyacrylamide gel (1mm thick). The IPG strip was placed on the SDS polyacrylamide gel and sealed with 0.5 % (w/v) agarose overlay. The gels were then placed in the electrophoresis tank filled with Laemmli running buffer (1 g/l SDS, 14.4 g/l glycine, and 3 g/l Tris pH 8.3) run at a constant voltage of 200V. The electrophoresis was stopped before the bromophenol blue dye reached the lower level of the gel. The gels were stained with Coomassie blue and scanned with GS-800 Calibrated Densitometer (Bio-Rad Laboratories, Hertfordshire, UK) to obtain 16-bit grayscale pictures.

2.8.2 Coomassie blue staining

After electrophoresis, the gels were gently removed from the glass slab, transferred into a suitable container, and washed briefly with distilled water to remove the remaining buffer. Gels were initially stained with Coomassie blue 0.1% (w/v) in fixative solution (40% (v/v) methanol and 10% (v/v) acetic acid. Destaining was achieved using several changes of destaining solution (20% (v/v) methanol and 7% (v/v) acetic acid.

2.8.3 Image analysis

The gel images were analysed using the ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, 1997-2014).The 2D gels were examined with the aid of suitable ImageJ plugins, following a protocol as described by Natale (2011); including bUnwarpJ and Watershed. The earlier plugin was used to align the images with a reference image, in order to remove the spatial distortion of the images resulting from dye-front deformation and run time differences, whereas the latter plugin performed the spot detection on the images. All data was normalised and statistically analysed by Anova test.

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2.8.4 Label-free protein quantitation and proteomics analysis

Label-free quantitative proteomics analysis is a proteomics platform based on the observation that the intensity of the MS spectrum is linearly proportional to the concentration of the ion being detected. (Levin et al., 2007) .

The isolated platelet proteins samples (section 2.5.3) were quantified and the concentrations were adjusted to obtain an equal amount of protein between all samples. To perform tryptic digestion, 50 µl of each sample was washed using 0.05% (w/v) RapiGest washing solution (Waters Corporation, USA) in 50 nM ammonium bicarbonate. Subsequently, the samples were reduced using 100 mM dithiothreitol (DTT) at 60°C for 15 minutes and alkylated at 25°C for 45 minutes in the presence of 200 mM iodoacetamide. Proteolytic digestion was performed overnight, using sequencing grade trypsin (Promega, USA) at a ratio of 1:50 (w/w) in a 37°C incubator. The digested proteins were hydrolysed by the addition of TFA at 37°C, before being vortexed and subsequently centrifuged at 13.000 rpm for 30 min.

The separation and subsequent mass spectrometry analysis of the tryptic peptides was performed according to the protocol described by Rodriguez-Suarez et al. (2013). Briefly, 1D nanoscale LC separation was performed with a nanoAcquity system (Waters Corporation, USA), employing a symmetry C18 5µm pre-column (20 cm x 180 µm) and a BEH C18 1.7 µm analytical RP column (20 cm x 75 µm). Mobile phase used were water (A) and acetonitrile (B) containing 0.1% (v/v) formic acid. After desalting and preconcentration, peptides were eluted from the pre-column to the analytical column and separated with a gradient of 3- 40% mobile phase B over 90 min at a flow rate of 300 nl/min, followed by a 10 min column

89 rinse with 90% mobile phase B at a flow rate of 300 nl/min and a constant temperature of 35°C.

Mass spectrometry analysis of the complex peptide mixtures was performed using SYNAPT G2-S HDMS mass spectrometer (Waters, Manchester UK), operated in a data-independent manner coupled with ion mobility (HDMSE). The analysis was performed in electrospray ionization mode with nominal resolution >20,000 FWHM. The experiment was programmed to step between low energy (4 eV) and elevated (14-40 eV) collision energies on the Triwave collision cell, using a scan time of 0.9 s per function over 50-2000 m/z. All samples were analysed in triplicate. Alignment of precursor and product ions by drift and retention time aids peptide identification by assignment of product ions to parent ions during data processing and database searching.

Protein identifications and quantification information were obtained using PROGENESIS QI by searching UNIPROT human database. Datasets of protein were investigated to assess the biological role, molecular function, and cellular localization by the aid of PANTHER classification system (Mi et al., 2013). Finally, pathway analysis was conducted by the aid of Panther pathway analysis and Reactome analysis tools.

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Figure 2.2 – Workflow of the proteomics study

Quantitative proteomics study is performed using a label-free method. Digested platelet proteome is separated using nanoAcquity UPLC, followed by mass spectrometry analysis by SYNAPG2 and SYNAPT G2S HDMS. The resulting MS spectra is analysed using Progenesis QI for protein identification and quantification, followed by further overrepresentation and pathway analysis by PANTHER and REACTOME software.

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