*2.2. LC-MS/MS Identification of Non-Protein Substances Accompanying MLP*

We washed out the unbound small molecules and proteins with no affinity from the column (Figure 2, fractions 1–18). However, because we also aimed to identify nonprotein substances that might possibly be present in the nucleolytic fractions, we extended our toolbox by adopting sample preparation from plasma/serum pharmacokinetics (Figures S2–S33) and combining it with LC-MS/MS screening of small molecules. In the fractions derived from the whole plant extract, we found the following alkaloids: berberine, chelidonine, coptisine, corysamine, dihydroberberine, dihydrosanguinarine, dihydroxychelerythrine, dihydroxysanguinarine, norchelerythrine, norsanguinarine, and protopine (Figures S3–S21) [4,5]. For samples rich in low-molecular constituents, we semi-quantitatively compared 21 signals (Tables S4 and S5; Figures S34–S47). Finally, we identified 8 alkaloids co-localizing with proteins in the protein-rich fractions. The presence of these alkaloids suggest their molecular association with proteins. The highest relative signal intensities for coptisine, berberine, dihydroberberine, chelidonine, and protopine were found in fractions 22–23 (Figure 3, in green), while those for 8-hydroxycheleritrine, stylopine, and sanguinarine were found in fractions 25–26 (Figure 3, in green).

m.60714 m.60714 glyceraldehyde-3-phosphate

lopine, and sanguinarine were found in fractions 25–26 (Figure 3, in green).

m.37897 m.37897 MLP-like protein 28 537 22910 7 42.7 m.37895 m.37895 MLP-like protein 28 267 22792 5 36.2 m.61102 m.61102 pectinesterase 3-like 218 63766 4 8.8 m.12630 m.12630 MLP-like protein 28 174 16874 2 17.7

cytosolic-like 100 36972 1 4.1

We washed out the unbound small molecules and proteins with no affinity from the column (Figure 2, fractions 1–18). However, because we also aimed to identify nonprotein substances that might possibly be present in the nucleolytic fractions, we extended our toolbox by adopting sample preparation from plasma/serum pharmacokinetics (Figures S2–S33) and combining it with LC-MS/MS screening of small molecules. In the fractions derived from the whole plant extract, we found the following alkaloids: berberine, chelidonine, coptisine, corysamine, dihydroberberine, dihydrosanguinarine, dihydroxychelerythrine, dihydroxysanguinarine, norchelerythrine, norsanguinarine, and protopine (Figures S3–S21) [4,5]. For samples rich in low-molecular constituents, we semi-quantitatively compared 21 signals (Tables S4 and S5; Figures S34–S47). Finally, we identified 8 alkaloids co-localizing with proteins in the protein-rich fractions. The presence of these alkaloids suggest their molecular association with proteins. The highest relative signal intensities for coptisine, berberine, dihydroberberine, chelidonine, and protopine were found in fractions 22–23 (Figure 3, in green), while those for 8-hydroxycheleritrine, sty-

*2.2. LC-MS/MS Identification of Non-Protein Substances Accompanying MLP* 


**Figure 3.** Correlation of the protein content (MLP) with the alkaloid content of the fraction. Top: Chromatogram of *C. majus* latex protein fractions adjusted to the numbers of fractions in the table. Blue line indicates protein content in fraction (monitored at 280 nm). The protein peak between fractions nos. 19–26 presents higher protein content in the fractions, which could be correlated with the higher quantity of identified alkaloids in the same fractions. Bottom: Relative signal intensities for latex fractions. Color coding ranges from red (lowest relative intensity of a particular compound in a group **Figure 3.** Correlation of the protein content (MLP) with the alkaloid content of the fraction. Top: Chromatogram of *C. majus* latex protein fractions adjusted to the numbers of fractions in the table. Blue line indicates protein content in fraction (monitored at 280 nm). The protein peak between fractions nos. 19–26 presents higher protein content in the fractions, which could be correlated with the higher quantity of identified alkaloids in the same fractions. Bottom: Relative signal intensities for latex fractions. Color coding ranges from red (lowest relative intensity of a particular compound in a group of samples) up to green (highest relative intensity).

> We used the same approach for *C. majus* milky sap, which enabled to identify several alkaloids (Table S5). Although the proposed identifications were based only on mass spectrometry data, they were consistent with the compounds detected previously in *C. majus* [4,5].
