Figure 5.

MS/MS findings verification. (A,B) Parallel reaction monitoring (PRM) verification of a peptide deriving from Outer Surface Protein A (OspA). OspA peptide AVEIKTLDELK was found in the discovery phase and confirmed by PRM in 2 non acute patient samples. (C–E) The presence of OspC (samples 214378, 108369, 243325), OspA (samples 108319, 881776) and Flagellin (sample 109126) was confirmed in by means of western blot analysis (MW molecular weight, PC positive control, NC negative control). Positive bands were found at 31 kDa, 27 kDa, and 18 kDa for OspA, OspC and Flagellin respectively. Since the positive control used in the western blot for flagellin is a flagellin–maltose-binding protein fusion protein, multiple bands above 40 kDa were detected (right image, PC lane). Patient 109126 yielded a band reactive to the anti-flagellin antibody at a lower molecular weight than the expected band at 34 kDa for the full length protein. This might be due to a truncated form of the protein. Uncropped original images are reported in Figure S16. (F,G) Western blot analysis detected the presence of Babesia BmSA1 and BMR1_03g00947 antigens in patients 542019, 413743, and 908230 who had at least one Babesia peptide in their urinary peptidome analysis conducted with mass spectrometry (2, 3, and 1, respectively). Patients 891284, 991873, and 243325 who had 2, 1, and 1 Babesia mass spectrometry peptide, respectively, did not show clear reactivity towards these two antibodies. Bands at a molecular weigth lower than the full length protein (48.7 kDa and 35.4 kDa for BMR1_03g00947 and BmSA1, respectively) are indicative of degradation products that pass glomerular filtration. PC is Babesia infected hamster red blood cells (RBC) diluted in urine. NC is healthy donor urine. PC − NP and PC + NP is positive control in absence and presence of nanoparticle processing, respectively. (H) Babesia peptides were identified in the blood and urine of a hamster animal model at early³⁶ and late stages of disease with high sensitivity. (I,J) Verification of MS analysis results of two surface/secreted B. microti antigens, BmSA1 and BMR1_03g00947, by Western blotting of hamster RBC lysates.