*4.6. MALDI-TOF MS Analysis*

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.

#### *4.7. Inequivocal Identification of Ricin in the Samples*

The identification of ricin in the samples was done through MALDI-TOF MS, using the PMF technique, after digestion of RTA and RTB.

#### 4.7.1. SDS-PAGE Under Reducing Conditions

SDS-PAGE of gels were made in an equipment Loccus, model LP3000. The racing and stacking gels were with 12% and 5% ( *w*/*v*) of polyacrylamide, respectively. For each sample a 20 mg/mL PBS10 bu ffer solution (pH 7.4) was prepared. This solution (4 μL) was added to 8 μL of a charging bu ffer containing the colorant bromophenol blue and other reactants (the commercial product "Blue Loading Bu ffer Pack", from BioLabs was used, following instructions of the manufacturer with the addition of the reducing agen<sup>t</sup> dithiothreitol). Finally, 10 μL of the charging solution was applied in one of the spots of a polyacrylamide gel. The bands were removed from the SDS-PAGE gel and identified through MALDI-TOF/MS.

The electrophoresis experiments were performed with a constant tension of 200 V until the migration line achieve 1 cm from the bottom. The gel was immersed in a solution containing ethanol/glacial acetic acid/water (45:10:45) and 1 g of the colorant Coomassie blue for 30 min under gentle stirring. The revelation was done overnight with a solution of methanol/glacial acetic acid/water (3:1:6) at room temperature. Solvents and reagents used were: bright Coomassie blue R250, 98.5%, from Vetec (Rio de Janeiro, Brazil); ethanol 95%, PA, from ACS, Isofar (Duque de Caxias, Rio de Janeiro, Brazil); glacial acetic acid 99.8%, PA, from ACS, Proquímios; methanol 100%, from ACS, J.T. Baker; and distilled and deionized water produced in the lab.
