Sinapinic acid

Matrix was prepared by dissolving 10 mg of sinapinic acid (Sigma, Germany) in 1.0 ml of 50% acetonitrile, 50% proteomics grade water, and 0.1% trifluoroacetic acid (TFA). Dried IP eluates were resuspended in 0.1% TFA and mixed with the matrix in a ratio of 1:1. 1.5 µl of each sample was deposited on the MALDI plate and allowed to cocrystallize at room temperature. Spectra were calibrated using readymade peptide calibration standard (Bruker Daltonics, USA). Peaks were acquired using repiflex MALDI Tissuetyper (Bruker Daltonics, USA) in a m/z range of 2000 to 6000 using positive linear mode. Five measurements were taken for each sample and the average spectrum was generated. Peaks were analyzed in FlexAnalysis (Version 3.4, Bruker Daltonics, USA) and Aβ proteoforms were manually annotated based on m/z values. Proteoforms with a signal/noise ratio ≥ 2 and deviation of no more than 5 Da from theoretical mass were included in the analysis. Proteoforms that were detected in at least two out of three independent replicates were included in the final dataset.

The extracted protein was spotted onto a steel target plate using a double-layer sinapinic acid (SA) method [37 (link)]. The first layer was composed of 0.7 μL of SA-saturated solution (10 mg/mL SA in absolute ethanol) (sinapinic acid from Sigma-Aldrich, Missouri, EUA). For the second layer, protein extract was mixed 1:1 with 10 mg/mL of SA solution in acetonitrile (30:70 v/v) (Merck, Darmstadt, Germany) and 0.1% trifluoroacetic acid (Sigma-Aldrich) in water. One microliter of this sample/matrix mixture was deposited onto a spot containing the first layer. The sample was left to dry at room temperature and then analyzed by MALDI-TOF MS. Each sample was analyzed in triplicate, loaded once into three different spots (i.e., three spectra per sample).

Cyt c (from equine heart), phosphatidylcholine (eggPC, from egg yolk), 3-Mercaptopropionic acid, 6-Mercaptohexanol, potassium chloride (KCl), sodium l-ascorbate, ethanol, 1-Octadecanethiol (ODT), trifluoroacetic acid (TFA), acetonitrile (ACN), and sinapinic acid were purchased from Sigma (St. Louis, MO). The lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (18:1, CL) were obtained from Avanti Polar Lipids (Alabaster, AL). Sodium dodecyl sulfate (SDS) was purchased from Merck (Darmstadt, Germany). Deuterium oxide (D₂O, 99.9%) was obtained from Cambridge Isotope Laboratories (Tewksbury, MA). Immobilized pepsin on 6% agarose beads was purchased from Thermo Scientific (Waltham, MA). A protein assay dye reagent concentrate was purchased from Bio-Rad (Hercules, CA). All aqueous solutions were prepared using ultrapure water (18.2 MΩ cm) from a Milli-Q purification system (Millipore, Burlington, MA). A CL binding dye, 10-nonyl acridine orange (NAO), was obtained from Enzo Life Sciences (Farmingdale, NY).

The samples were analyzed in a MALDI-TOF mass spectrometer, Bruker^®, model microflex LRF. This equipment has a laser of N₂, with a maximum frequency of 60 Hz, and minimal focus of 50 µm. Each sample was analyzed in triplicate. Firstly, 0.5 µL of solution of sinapinic acid (99% from Sigma-Aldrich (São Paulo, Brazil), saturated in ethanol (95% PA from ACS, Isofar (Duque de Caxias, Rio de Janeiro, Brazil), were applied in one of the spots of a stainless steel target plate. This solution was left to dry and a thin layer of matrix was formed. After, a second solution was prepared, this time containing sinapinic acid saturated in TA30 [30% acetonitrile with 70% water/trifluoroacetic acid (99.9:0.1)]. This solution was mixed in equal parts with a third solution, containing 2 mg/mL of the original sample diluted in 0.1% TFA/water. One aliquot of 0.5 µL of this mixture was collected and applied over the first matrix layer described above, left to dry and analyzed.
The analyses were performed in linear mode by monitoring the presence of peaks in the spectral region corresponding to masses between 50 and 70 kDa. All spectra were acquired by addition after 3000 laser shots, randomly distributed over the whole surface of the sample. The laser energy was kept constant in all shots.

The multiphoton microscope with a Chameleon Ultra II tuneable laser (690–1080 nm range excitation, Coherent, Santa Clara, CA, USA) enables the excitation of secondary metabolites in a manner similar to a UV laser (Conéjéro et al., 2014 (link); Talamond et al., 2015 (link)). Optimal excitation was obtained at a wave length λ = 720 nm and band-pass emission in the 410–650 nm range using an array of 32 photomultiplier tube (PMT) detectors (Zeiss), each with an 8.8 nm bandwidth.
This spectral detector yielded spectral images and emission spectra from the epidermal and hypodermal walls of fresh root sections of Fo072 inoculated or control vanilla accessions. After obtaining the spectral acquisitions, the Linear Unmixing (ZEN software, Zeiss, Jena, Germany) function was executed to separate, pixel by pixel, the mixed signals of six defined pure autofluorescent compounds namely ferulic acid, conyferylic acid, sinapinic acid, p-coumaric acid, caffeic acid, and quinic acid (Sigma-Aldrich, St. Quentin Fallavier, France), using the entire emission spectrum of each compound plus a residual channel. This image analysis showed each compound present in the sample with coded colors. In the residual channel, the intensity values represented the difference between the acquired spectral data and the fitted linear combination of the reference spectra.