MALDI-TOF MS Technique as a New Approach for Simultaneous Detection and Differentiation of Potato Virus Y Strains

Both zipped folders contains raw data for figures presented in the manuscript "MALDI-TOF MS Technique as a New Approach for Simultaneous Detection and Differentiation of Potato Virus Y Strains" under review in Scientific Reports. Fig1_RawData.zip contains files with raw data for Figure 1 Fig2-11_MALDI-TOF Raw Data.zip contains excel files with raw data generated during MALDI-TOF experiments for Figures 2-11. Steps to reproduce data in Fig1_RawData.zip: Plant material - the source of PVY. PVY isolates – Bonin 1 (PVYO), Bonin 2 (PVYN-Wi), and Bonin 3 (PVYNTN) were maintained in potato leaves at -80°C. These isolates were multiplied in Nicotiana tabacum L, cultivar Samsun. The first set of tobacco plants (five plants per PVY isolate) was mechanically inoculated with sap extracted from 1g of deep-frozen leaves by grounding in liquid nitrogen with mortar and pestle, mixing with 3 ml of nuclease-free water, and pressing through the fabric to remove the solids. Two leaves of the tobacco plant (at the 3-4 leaves development stage) were dusted with carborundum, and 0.1 ml of virus-containing sap was rubbed into the mechanically damaged area. After ten minutes, the carborundum was washed off using ultrapure water. The plants were grown in a growth room under controlled light (LED Greenie T8 Flora, 70 μmol·m–2·s–1), temperature (21°C), and humidity (80%), with 16/8h day/night cycle. Plants were monitored for symptoms starting the fifth-day post-inoculation (dpi). When most leaves were symptomatic (usually between 10-14 dpi), the sap from leaves was extracted using Pollähne press (MEKU Erich Pollähne GmbH), and the progress of infection was monitored using the TAS-ELISA test (Agdia, cat. number: 20001) performed according to the manufacturer instruction. The optical density (OD) was measured after 30 of incubation of the substrate at room temperature (RT) at λ=405 nm using the BioTek Epoch microplate spectrophotometer (Agilent Technologies). Data were processed using BioTek Gen5 Software (Agilent Technologies). The leaves from plants with the highest OD values were collected, and sap was extracted using the Pollähne press. The extracted sap was used to inoculate the second set of tobacco plants dedicated to purifying virus preparations (twenty plants per PVY strain). The plants were monitored for virus titer as described above, and leaves with the highest viral titer were collected to purify pure preparations of PVY strains. Virus isolation. The pure preparations of Bonin 1-3 isolates representing three PVY strains (O, N-Wi, and NTN) were isolated from tobacco leaves. The leaves (0.5 kg per isolation) were homogenized in liquid nitrogen using a pestle and mortar. The resulting fine powder was mixed in a 1:1 (v/v) ratio with a cold 20 mM borate buffer, pH 8.0, containing 0.03% ascorbic acid, 0.3% 2-mercaptoethanol, 0.01 mM DEPC, 5 mM PMSF, and a cocktail of protease inhibitors (Complete, La Roche). Next, the slurry was filtered through the fabric and centrifuged at 10 000×g for 10 min at room temperature (RT). Chloroform was slowly added to the supernatant up to 10%. After 30 minutes of mixing, the layers were separated by centrifugation at 10 000×g for 10 minutes at RT. In the next step, Triton X-100 was added to the supernatant to a final concentration of 2%, and the mixture was stirred at room temperature for 15 minutes. Subsequently, NaCl was added to a final concentration of 2%, and PEG-6000 was added to 8%. The mixture was kept in ice and stirred for 1 h, followed by centrifugation at 10 000×g for 10 minutes at 4°C. The pellet was dissolved in ice-cold 20 mM borate buffer, pH 7.8, for 1 h, followed by centrifugation at 10 000×g for 10 minutes at 4°C. The supernatant was retained and stored at 4°C. The pellet was dissolved again in the same buffer overnight at 4°C. The next day, the undissolved pellet was centrifuged, and the supernatant was combined with previous portions. The clarified preparation was centrifuged at 10 000×g for 10 minutes at 4°C. The obtained supernatant was layered onto a cushion of 30% sucrose in 20 mM borate buffer and centrifuged for 6 hours at 80 000×g and 15°C. The resulting pellet was dissolved in 20 mM borate buffer, pH 7, overnight at 4°C. The following day, clarification was performed for 5 minutes at 3000×g and 15°C, and the supernatant was collected. The purity and concentration of the virions were determined spectrophotometrically on the Biotek Epoch spectrophotometer, assuming that for the pure virus, the OD ratio 260/280 nm equals approximately 1.21, and OD260 nm = 2.8-2.9 corresponds to 1 mg/ml PVY virions. The measurement of total protein in pure PVY preparations was performed according to Whitaker and Granum (1980) using the equation C[mg/mL] = (OD235nm - OD280nm) / 2.51, where C is the protein concentration, and 2.51 is the correction factor relating the OD difference to 1 mg of protein in one milliliter/ The relative content of viral coat protein was estimated using the TAS-ELISA test (Agdia, cat. number: 20001) performed according to the manufacturer's instructions, with the exception that OD readings at 405 nm were taken after a 10-minute incubation with p-nitrophenol phosphate [28]. Data were processed using BioTek Gen5 Software. The relative content of PVY cp in the obtained preparations was expressed as the ratio of the relative content of PVY coat protein to the total protein present in the preparation. Isolation of PVY genomic RNA. To isolate genomic RNA, 100 µL of the viral preparation (isolated following the procedure described above) was mixed with 900 µL of Trizol. The mixture was stirred, and the resulting emulsion was incubated for 10 minutes at RT. Subsequently, 0.2 mL of chloroform was added to the samples, vortexed for 30 seconds, and then incubated for 5 minutes at RT with rotary shaking on a rotator. The phases were separated by centrifugation for 15 minutes, 12 000×g, 4°C. The phenol phase (lower) was carefully separated from the aqueous phase (upper). To the tubes containing the aqueous phase, 8 µL of co-precipitant (dyed glycogen or linear polyacrylamide) was added and vortexed for 30 seconds. Then, 0.5 mL of isopropanol was added, and the mixture was thoroughly mixed by tube rotation, followed by incubation on ice for 10 minutes. The precipitate was centrifuged into a pellet for ten minutes at 18 000×g at 4°C. The supernatant was carefully removed. The RNA pellet was suspended in 1 mL of 75% ethanol solution to dissolve any remaining salts beneath the RNA pellet. It was then centrifuged to a pellet at 18000×g at 4°C, and the supernatant was discarded. The pellet was dried for 3 minutes at 56°C, re-suspended in 50 L of water heated to 56°C, and then incubated on ice for 30 minutes. The quality and quantity of the RNA were analyzed using the Take3 plate (Agilent Technologies) for microvolume detection on the Biotek Epoch spectrophotometer. OD values were measured for undiluted RNA preparations (2 µl) at 230, 260, and 280 nm, and the readings were corrected for light scatter by subtracting OD 320 nm. Data were processed using BioTek Gen5 Software. The RNA was qualified as pure when the 260/280 ratio was 1.8-2.1 and the 260/230 ratio - 2.0-2.2. For pure preparations of gRNA, the concentration was calculated assuming that for single-stranded RNA, OD260 = 1.0 corresponds to 40 µg/mL. Real-time RT-PCR analysis. To assess the relative genomic RNA content in the obtained PVY strain preparations (Fig. 1C), a tenfold dilution was performed using nuclease-free mQ water. Undiluted preparations could potentially contain excessively high RNA concentrations, obscuring any differences between the samples. Five such dilutions were prepared for each sample (technical replicates). From each tenfold-diluted sample, 3 µL of the preparation was added to the RT-qPCR reaction mixture. To detect PVY quantitatively, the Y1 set of primers and probe, specifically amplifying the region of PVY inside the coat protein coding ORF, was chosen. The reaction mixture contained the TaqPath™ 1-Step Multiplex Master Mix (Applied Biosystems™), Y1 forward and Y1 reverse primers (0.3 µM each), FAM-labeled Y1 probe (0.1 µM) and nuclease-free water. The final volume of the reaction mixture was 10 µL. 96-well microplates with reaction mixtures were sealed with foil, centrifuged for 30 seconds, and placed in the real-time PCR cycler, CFX Touch 96-well Real-Time PCR Detection System (Bio-Rad Laboratories). Amplifications were conducted following a thermal profile: 25°C - 2 min (activation of uracil N-glycosylase), 53°C - 10 min (reverse transcription – conversion of RNA to cDNA), 95°C – 2 min (initial denaturation of cDNA). After the initial denaturation, 40 cycles were programmed, consisting of repeated stages of DNA denaturation - 95°C for 15 seconds and extension of new DNA strands by the polymerase at 60°C for 1 minute. Fluorescence readings were taken after each extension stage. The data were analyzed in CFX Maestro 1.1 software (Bio-Rad Laboratories). Primers and probe used for Real-time RT-PCR analysis: Y1 FP CCA ATC GTT GAG AAT GCA AAA CY1 RP ATA TAC GCT TCT GCA ACA TCT GAG AY1 Probe FAM-TTA GGC AAA TCA TGG CAC AT-BHQ1 Isolation of coat protein (CP) from the PVY pure preparations. For PVY cp isolation, 100 µL of the viral preparation was mixed with 900 µL Trizol. The mixture was stirred, and the resulting emulsion was incubated for 10 minutes at RT. Subsequently, 0.2 mL of chloroform was added to the samples, vortexed for 30 seconds, and then incubated for 5 minutes at RT with rotary shaking on a rotator. The phases were separated by centrifugation for 15 minutes at 12000×g at 4°C. The phenol phase (lower) was carefully separated from the aqueous phase (upper). To the tubes containing the phenol phase, 0.3 mL of 98% ethanol was added and thoroughly mixed on a rotator for 5 minutes at RT. Then, the deoxyribonucleic acid (DNA) was precipitated by centrifugation for 5 minutes at 2000×g at 4°C. The phenol-ethanol supernatant was transferred to a new tube with a volume of 2 mL. 1.5 mL of isopropanol was added, and incubation was done for 10 minutes. The protein pellet was precipitated by centrifugation for 10 minutes at 18000×g at 4°C. The supernatant was carefully removed. The protein pellet was dissolved in 2 mL of a 0.3 M guanidine hydrochloride solution in 95% ethanol. Incubation was done for 20 minutes at RT. The protein was precipitated by centrifugation for ten minutes at 18000×g at 4°C, and the supernatant was discarded. The guanidine hydrochloride wash of the protein was repeated twice, followed by washing with two milliliters of 98% ethanol. The protein suspended in ethanol was vortexed for 30 seconds, incubated for 30 min at RT, and centrifuged for 5 minutes at 7500×g at 4°C. The supernatant was carefully removed and discarded. The protein pellet was air-dried for 10 minutes, then dissolved in 50 µL of a 1% SDS solution. An equal volume of 1 M Tris-HCl buffer at pH 8.0 with 8 M urea was added, resulting in a final composition of 0.5% SDS, 0.5 M Tris-HCl buffer, and 4 M urea in the preparation. After dissolution, centrifugation was done for 10 minutes at 10000×g at 4°C. The supernatant (protein) was transferred to a new tube. Total protein concentration was measured using a Qubit Protein Assay Kit and fluorescence reading on a Qubit Flex fluorimeter (Thermo Fisher Scientific, cat number: Q33212). SDS-PAGE electrophoresis. The protein composition of purified PVY strain preparations was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and denaturing conditions in Laemmli buffer system. Electrophoresis was performed in a Mini-PROTEAN Tetra cell apparatus (Bio-Rad Laboratories) using a gradient polyacrylamide 4-15% TGX Stain-Free™ Protein Gels (w/v) (Bio-Rad Laboratories). Viral preparations were adjusted to a concentration of 20 µg of viral particles in 100 µL of a sample containing, in addition to the virus, 125 mM Tris-Cl at pH 6.8, 0.1% SDS, 100 mM dithiothreitol (DTT), 10% (v/v) glycerol, and 0.1% (w/v) bromophenol blue. The samples were denatured for 5 minutes at 95°C, then immediately cooled on ice for 5 minutes and centrifuged at 18 000×g for 15 minutes at 4°C. The supernatant from the prepared samples (30 µL each) was applied to the polyacrylamide gel, i.e., 6 µg of viral particles were loaded on each lane. Electrophoresis was performed at 100 V for 1 hour at 4°C. After completion of the separation, the gels were documented using the ChemiDoc MP System (Bio-Rad Laboratories) with stain-free detection settings. The Kang et al. (2002) staining was applied for permanent records. Gels were denatured for 15 minutes in a solution of 30% (v/v) ethanol, 10% (v/v) methanol in 2% (v/v) phosphoric acid, and then stained for 2 hours with a colloidal solution of Coomassie Blue - 0.02% (w/v) Coomassie Blue G-250 in 10% (v/v) ethanol, 5% (w/v) aluminium sulfate 18-hydrate, and 2% (w/v) phosphoric acid. The gels were destained with 10% ethanol in 2% phosphoric acid, changing the solution every few hours until the background was destained entirely [29]. The gels were documented using the ChemiDoc MP with settings and filters for colorimetric documentation. Steps to reproduce data in Fig2-11_MALDI-TOF Raw Data.zip: Mass spectrometry analysis. In the first step of virus analysis by MALDI-TOF the method was validated. Viruses, RNA preparations, and protein preparations of PVY (O, NTN, N-Wi strains) were analyzed. MALDI measurements were performed on the Bruker 384 polish steel target. The matrices for MALDI analysis HCCA (α-Cyano-4-hydroxy-cinnaminic acid) and DHB (2,5-dihydroksybenzoic acid, Bruker Daltonik GmbH, Bremen, Germany), were prepared as follow - (1) 10mg/mL HCCA saturated in TA30 (30:70 ACN:0.1% TFA in water v/v, (2) 10mg/mL HCCA saturated in TA50, (3)10 mg/mL DHB in TA30, and (4) 10 mg/mL DHB in TA50 (50:50 ACN:0.1% TFA in water v/v). 1 μL of each sample was placed directly on the target and air-dried, then 1 μL of the matrix was applied to the spots. MS experiments were performed using a Bruker ultrafleXtreme II time-of-flight mass spectrometer equipped with a SmartBeam II laser (355 nm, frequency 2 kHz) in positive ion reflectron (RP) and linear modes (LP). The measurement range was m/z (1)0-2000, (2)2000-20000, (3)20000-100000. Laser power was 30%, 45%, 60%, 75%, and 100%. For ultrafleXtreme II in RP mode, the following parameters are applied: laser- global attenuators offset 50%, attenuator offset 17%, attenuator range 33%, focus offset 0%, focus range 100%, Focus position 9%, digitizer- sensitivity 100 mV, analog offset linear 2.0 mV, analog offset refceltor2.6 mV, trigger level 800 mV, detector gain voltages 2600V, reflector base 1800V, spectrometer- ion sources 1 25.07kV, ion sources 2 22.48kV, lens 8.18 kV, pulsed ion extraction 110 ns. For ultrafleXtreme II in LP mode, the following parameters are applied: laser- global attenuators offset 20%, attenuator offset 20%, attenuator range 40%, focus offset 0%, focus range 100%, focus position 9%, digitizer- sensitivity 100 mV, analog offset linear 2.0 mV, analog offset reflectron 2.6 mV, trigger level 800 mV, detector gain voltages 2600V, reflector base 1800V, spectrometer- ion sources 1 25.07kV, ion sources 2 23.27kV, lens 7.53 kV, pulsed ion extraction 400 ns. The number of laser shots was 5000 (10 × 500 shots) for each sample spot. Mass calibration was performed with FlexAnalysis 3.3 using the cubic/quadratic enhanced model and standards- Cs clusters; BTS standard, Protein Standard II (Bruker Daltonik GmbH, Bremen, Germany). For the dilution series experiment, three independent virus preparations were used as biological replicates for each PVY strain (O, NTN, N-Wi). For each dilution point (stock, 10⁻¹, 10⁻², 10⁻³), three technical replicates were acquired by spotting the same viral preparation on three distinct MALDI target positions. Thus, each dilution level was represented by nine spectra per virus strain (3 biological × 3 technical replicates), which were used for statistical analysis. MALDI measurements were performed on the MBT Biotarget 96. The matrices for MALDI analysis HCCA and DHB were prepared as follows—(1) 10mg/mL HCCA saturated in TA30 (30:70 ACN:0.1% TFA in water, (2) 10mg/mL HCCA saturated in TA50, (3)10 mg/mL DHB in TA30, and (4) 10 mg/mL DHB in TA50 (50:50 ACN:0.1% TFA in water). An amount of 1 μL of each sample was placed directly on the target and air-dried, then 1 μL of the matrix was applied to the spots. MS experiments were performed using a Bruker microflex. The measurement range was m/z 2000-20000. Laser power was 60%. For microflex, the following parameters are applied: laser- global attenuators offset 0%, attenuator offset 21%, attenuator range 30%, digitizer- sensitivity 100 mV, analog off set linear -1.0 mV, analog offset reflector 0.0 mV, trigger level 800 mV, detector gain voltages 2500V, reflector base 1400V, spectrometer- ion sources 1 25.07kV, ion sources 2 22.48kV, lens 8.18 kV, pulsed ion extraction 110 ns. spectrometer - ion sources 1 20.03kV, ion sources 2 18.02kV, lens 6.07 kV, pulsed ion extraction 120 ns. The number of laser shots was 5000 (10 × 500 shots) for each sample spot. Mass calibration was performed using FlexAnalysis 3.3 software (Bruker Daltonics GmbH) with the enhanced cubic/quadratic calibration model, utilizing the Bruker Test Standard (BTS) as an external calibrant. During data preprocessing and peak picking, only signals with a signal-to-noise (S/N) ratio ≥ 3:1 were considered as valid peaks. The local noise level was estimated using a sliding window approach implemented in the software to ensure accurate baseline subtraction. This threshold minimized the inclusion of low-intensity, noise-associated signals and ensured that only analytically relevant ion peaks were subjected to further statistical analysis. Data analysis. Results obtained during virions, proteins, and gRNA isolation and from RT-qPCR were analyzed using GraphPad Prism 10 (GraphPad Software). MS data analysis was performed in the R environment, using RStudio 2023.06.1 console (PBC, Boston, MA, USA). Statistical methods were performed using “stats” functions. The normality of data distribution was assessed using the Shapiro-Wilk test. Mann−Whitney test was applied to verify statistically relevant ion intensities (m/z) differences across the virus species (whole virus, protein, and RNA extracts), analyzed under various analytical conditions. A significance criterion of p < 0.05 was considered. Principal component analysis (PCA) was performed on the scaled data (z-score normalization) using the packages “factoextra” and “Facto-MineR.” Random forest algorithm (RF) was implemented using the “caret” package. The calibration and validation steps used 2/3 and 1/3 of the available data, respectively. Model calibration was performed employing a 10-fold cross-validation, in three repetitions (“repeatedcv” method). The number of variables randomly sampled as candidates at each split was optimized for each model during the calibration step. The number of trees was set as 500 for all models. Graphs were prepared employing “ggplot2” functions.