*2.5. Detection of Oxidized Proteins (OXs) by Resin-Assisted Capture RAC (OX–RAC)*

The detection of OXs by OX–RAC was performed on three biological replicates using a previously described protocol [6]. Captured proteins were eluted with 30-μL elution buffer containing 10 mM HEPES, 0.1 mM EDTA, 0.01 mM neocuproine, 0.1% sodium dodecyl sulfate (SDS), and 100 mM 2-mercaptoethanol for 20 min at room temperature. Proteins in a 10-μL aliquot of each eluent were resolved on a 12.5% SDS—polyacrylamide gel electrophoresis (PAGE) gel. Each gel was then stained with silver (Pierce Silver Stain), and each gel slice was independently analyzed by MS. A protein was considered to be oxidized when its relative amount in the DTT-treated lysates was significantly more than that in the DTT-untreated lysates (*p <* 0.05 according to the results of an unpaired *t*-test).

## *2.6. In-Gel Proteolysis and MS Analysis*

The proteins in the gel were reduced with 2.8mM DTT (60 ◦C for 30 min), modified with 8.8 mM iodoacetamide in 100 mM ammonium bicarbonate (in the dark and at room temperature for 30 min) and digested in 10% acetonitrile and 10 mM ammonium bicarbonate with modified trypsin (Promega, Beit Haemek, Israel) overnight at 37 ◦C. A second trypsin digestion was carried out for another 4 h at 37 ◦C.

The tryptic peptides were desalted using C18 tips (Home-made, 3M) dried and resuspended in 0.1% formic acid.

The peptides were resolved by reverse-phase chromatography on 0.075 × 180-mm fused silica capillaries (JW) packed with Reprosil reversed phase material (Dr Maisch GmbH, Ammerbuch, Germany). The peptides were eluted with linear 60 min gradient of 5 to 28% 15 min gradient of 28 to 95% and 25 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.15 μL/min. MS was performed by Q Exactive HF mass spectrometer (Thermo Fisher Scientific represented by BARGAL analytical instruments, Shoham, Israel) in a positive mode using a repetitively full MS scan followed by collision induces dissociation (HCD) of the 18 most dominant ions selected from the first MS scan. The mass spectrometry data were analyzed using the MaxQuant software 1.5.2.8, The Max Plank Institute of Biochemistry, Munich, Germany [24] vs. *Entamoeba histolytica* and *Lactobacillus acidophilus* proteomes from the Uniprot database with 1% FDR (false discovery rate). The data were quantified by label free analysis using the same software. Statistical analysis of the identification and quantization results was done using Perseus 1.6.7.0 software, The Max Plank Institute of Biochemistry, Munich, Germany [25]. A *t*-test between the groups with or without DTT was carried out, with the Benjamini–Hochberg correction for multiple testing. Proteins were considered as significantly changed if their *p*-value < 0.05, *q*-value < 0.05, and the fold change between the groups ≥ 1.
