*3.3. Difference in Protein Abundance Is Observed between NO-Resistant and NO-Susceptible L. braziliensis Strains and it Is Significantly Modulated in Response to NO*

As we observed that the NO-resistant strain was significantly more resistant to NO and more infectious for the host cells even after treatment with H2O2 and NaNO2, we decided to evaluate the protein abundance profile of this strain with and without NaNO2 challenge and compare it to the profile of the NO-susceptible strain. Mass spectrometry analysis of four biological replicates (independent biological assays) of each strain, challenged or not with 1/5 IC50/4 h NaNO2, allows for the identification of 6296 protein groups, encompassing ~80% of the *Leishmania* predicted proteome (~8000 protein-coding genes predicted, considering one protein per gene) (Table S1). More than 5700 protein groups were identified in each replicate, and 5010 protein groups were identified in all 16 samples (Table S2).

The total protein contents per cell were calculated as previously described [25] using the histone ruler method based on the DNA content reported for *L. braziliensis* reference (strain M2904). Similar to the estimates previously reported [25,33], the 2853 strain contains 3.3 ± 0.11 pg of protein per cell. Remarkably, the protein content of this strain increased significantly when parasites were challenged with the NO donor, reaching 4.3 ± 0.08 pg of protein per parasite (Figure 3A). Interestingly, the 2856 strain exhibited 4.4 ± 0.2 pg of total protein per cell, and this value did not change after challenge with NO (4.4 ± 0.1 pg) (Figure 3A).

Using the total protein approach (TPA) method, we calculated the absolute protein concentrations for each strain. In agreement with previous reports [25], we observed that protein concentration values span 6 orders of magnitude; 90% of the proteome extends over ~3 orders of abundance; and histones, alpha- and beta-tubulin, elongation factor 1-alpha, HSP70, and calmodulin are among the top 20 most abundant proteins (Figure 3B). Remarkably, principal component analysis (PCA) of the protein concentrations revealed a consistent separation between the NO-resistant and NO-susceptible strains, as well as between these strains after the NO challenge (2853+NO and 2856+NO) (Figure 3C), showing a clear clustering for each set of biological replicates. Statistical analysis by PCA also showed that the proteome of the NO-resistant strain is clearly modulated, in terms of protein concentrations, after the NaNO2 challenge.

Using Perseus, a total of 6022 proteins were statistically validated in at least 12 out of the 16 samples (FDR 0.01). The statistical significance of differences in protein abundance among the strains was determined by Student's *t* test at FDR of 3%. In total, the concentrations of 1320 proteins were significantly different between the NO-resistant strain and the NO-susceptible strain; the concentration of 474 proteins was different between these strains treated with NaNO2 (2853+NO and 2856+NO), 850 between 2853 and 2853+NO, and 122 between 2856 and 2856+NO (Table S3 and Figure S2). These results reveal natural intrinsic differences in the proteome between the resistant and susceptible strains and show that the NO-resistant strain more actively modulates its proteome in response to the NO challenge than the NO-susceptible one.
