2. Material and Methods
2.1 Sample preparation
In the study, a total of 10 vein DBS slices were analysed, with one slice taken from each subject that was conserved at room temperature for 3 months. During sample preparation, the DBS slices were perforated and crushed. A 2-mL Eppendorf tube containing a blood slice cut into small pieces was then prepared. To this tube, 250 μl of an extraction solution consisting of 50 mM ammonium bicarbonate, 2% SDS (sodium dodecyl sulphate), and 2% sodium deoxycholate (DOC) was added.
The prepared solution was then incubated at 37 °C for 45 min with stirring at 1400 rpm in a thermoblock to facilitate protein extraction and solubilization. After incubation, the sample was spun briefly for 15 seconds to sediment the crushed DBS discs. The supernatant containing the extracted proteins was carefully transferred to a conical tube.
For further analysis, the supernatant was divided into several aliquots. These aliquots were likely used for various downstream proteomic analyses, such as protein identification and quantification by mass spectrometry. Splitting the supernatant into multiple aliquots allowed parallel analyses or sample storage for later experiments. The characteristics of the study population are summarized in Table 1. In this work, 10 samples were used (5 CAD patients and 5 control patients). The parameters: weight, height, Tc, hdl cholesterol, ldl cholesterol, hct and plaq showed significant differences between the two groups (p < 0.05). In terms of Age, no significant differences were found between the two groups (p > 0.05).
2.2 LC-MS/MS analysis
The whole sample was diluted and protein concentration was determined using the Lunatic UV/Vis polychromatic spectrophotometer (Unchained Labs). Proteins were reduced with 2 mM TCEP (tris(2-carboxyethyl)phosphine) and alkylated with 15 mM Iodoacetamide at 30°C for 30 min. Samples were further processed using the single‐pot solid‐phase enhanced sample preparation (SP3). SP3 protein purification, digest and peptide clean-up were performed using a KingFisher Flex System (Thermo Fisher Scientific) and Carboxylate-Modified Magnetic Particles (GE Life Sciences; GE65152105050250, GE45152105050250) [AA]. Beads were conditioned following the manufacturer’s instructions, consisting of 3 washes with water at a concentration of 1 µg/µl. Samples were diluted with 100% ethanol to a final concentration of 50% ethanol. Beads, wash solutions and samples were loaded into 96-well deep-well plates and transferred to the KingFisher. Following steps were carried out on the robot: collection of beads from the last wash, protein binding to beads, washing of beads in wash solutions 1-3 (80% ethanol), protein digestion (overnight at 37°C with a trypsin-protein ratio of 1:50 in 50 mM Triethylammoniumbicarbonat (TEAB)) and peptide elution from the magnetic beads using MilliQ water. The eluted samples were dried to completeness and re-solubilized in MS sample buffer (3% acetonitrile, 0.1% formic acid) before measurement of peptide content and dilution to a final absorbance of 0.06 at 280 nm.
Mass spectrometry analysis was performed on an Orbitrap Exploris 480 (Thermo Scientific) equipped with a Nanospray Flex Source and coupled to an M-Class UPLC (Waters). Solvent composition of the two channels was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% acetonitrile for channel B. For each sample, 5 μL of peptides were loaded on a commercial MZ Symmetry C18 Trap Column (100 Å, 5 µm, 180 µm x 20 mm, Waters) followed by a nanoEase MZ C18 HSS T3 Column (100Å, 1.8 µm, 75 µm x 250 mm, Waters). The peptides were eluted at a flow rate of 300 nL/min. After an initial hold at 5% B for 3 min, a gradient from 5 to 35% B in 90 min was applied. The column was washed with 95% B for 5 min and afterwards the column was re-equilibrated to starting conditions for another 10 min.
Samples were acquired in a randomized order within each tissue. The mass spectrometer was operated in data-dependent mode (DDA) with a maximum cycle time of 3 s, using Xcalibur (Tune version 2.0), with spray voltage set to 2.0 kV, funnel RF level at 40 %, and heated capillary temperature at 275 °C. Full-scan MS spectra (300−1’200 m/z) were acquired at a resolution of 120’000 at 200 m/z after accumulation to a target value of 3’000’000 or for a maximum injection time of 45 ms. Data-dependent MS/MS were recorded in the linear ion trap using a quadrupole isolation with a window of 1.2 Da and HCD fragmentation with 30 % fragmentation energy. MS2 spectra were acquired at a resolution of 45’000 after fragmentation with an NCE of 30% using a maximum injection time set to auto and an AGC target of 100’000. Only precursors with intensity above 5’000 were selected for MS/MS. Charge state screening was enabled. Singly, unassigned, and charge states higher than six were rejected. Precursor masses previously selected for MS/MS measurement were excluded from further selection for 20 s, and the exclusion window was set at 10 ppm. The samples were acquired using internal lock mass calibration on m/z 445.1200.
2.3. Peptide and Protein Identification through Novel Sequence Analysis and Database Search
The data obtained were then processed using the software PEAKS, version X PRO (Biofinformatics Solutions Inc, Waterloo, Canada) using a precursor and fragment mass tolerance of 10 ppm and 0.025 Da, respectively. The peptide false discovery rate (FDR) was predictable by the Decoy fusion method and was set at a maximum of 1%. Peptides with a ratio ≥ 1.5 (overexpressed) or ≤0.67 (underexpressed) were considered to have different frequencies. Further refinement was then performed by filtering out the p value from a paired Student’s t test < 0.05. Perseus software 1.5.1.5 (provided free of charge by the Max Planck Institute, Munich, Germany) was used for the differential protein abundance analyses. Principal component analysis (PCA) and heat maps for data visualisation were performed with Metaboanalyst (www.metaboanalyst.ca). Mass spectrometry proteomics data were deposited with the ProteomeXchange Consortium via the PRIDE partner repository with dataset identifier PXD042493.