EhPKMT2-interacting proteins
Description
Hypothesis. The protein lysine methyltransferase 2 (EhPKMT2) regulates several cellular/molecular essencial processes of Entamoeba histolytica. Tse proteins were identified by pulldown assays usin the recombinant EhPKMT2 as bait. The results indicate that this PKMT is involved in transcription regulation, translation, metabolism, stress response and cytoskeleton dynamics, suggesting that this protein coul be an atractive target to develop new therapeutic strategies against amebiasis
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We performed pulldown assays using recombinant EhPKMT2 as bait and protein extracts of Entamoeba histolytica as prey. Proteins were analyzed by mass spetrometry sing a Synapt G2-Si mass spectrometer (Waters Corp). Tryptic peptides (4.5 µL) were injected and separated on a HSS T3 C18 Column (75 μm × 150 mm, 100 Å pore size, 1.8 μm particle size; Waters Corp) using UPLC ACQUITY M-Class (Waters Corporation). As mobile phases were used 0.1% FA in H2O (phase A) and 0.1% of FA in ACN (phase B) under the following gradient: 7% B (0 min), 40% B (121.49 min), 85% B (123.15-126.46 min) and 7% B (129-130 min), at a flow of 400 nL·min-1 at 45 ◦C. The spectra data were acquired using nano electrospray ionization and ion mobility separation (IM-MS) using Full-Scan DIA-approach through High-Definition Multiplexed MS/MS (HDMSE) mode (Lou and Shui, 2024). Parameters for the ionization source were set at 2.75 kV in the sampling capillary, 30 V in the sampling cone, 30 V in the source offset, 70 °C for the source temperature, 0.5 bar for the nanoflow gas and 150 L·h−1 for the purge gas flow. Two chromatograms were acquired (low and high energy chromatograms) in positive mode in a range of m/z 50−2000 with a scan time of 500 ms. For the high-energy chromatograms, the precursor ions were fragmented in the transfer cell using a collision-induced dissociation (CID) energy ramp from 19 to 55 eV. Calibration of the Synapt G2-Si was performed with [Glu1]-Fibrinopeptide fragments via the precursor ion [M+ 2H]2+ = 785.84261, with a fragmentation of 32 eV and an error of 1.3 ppm in all MS/MS measurements. Database Search The *.raw files containing MS and MS/MS spectra were analyzed with the software Protein Lynx Global Server (PLGS) v4.2 (Waters Corporation, Milford, MA, USA) (Li et al., 2009) using a target decoy strategy to detect false positives (Käll et al., 2008) against a concatenate *.fasta file of Entamoeba histolytica (downloaded from Amoeba DB, release 68). Parameters used during the database search were set as follows: trypsin as a cut enzyme and one missed cleavage allowed; carbamidomethyl (C) as a fixed modification and amidation (N-term), deamidation (N, Q), oxidation (M), phosphoryl (S, T, Y) as variable modifications; automatic peptide and fragment tolerance, minimum fragment ion matches per peptide: 2, minimum fragment ion matches per protein: 5, minimum peptide matches per protein: 1, and a false discovery rate (FDR) ≤1 %. Quantification of proteins was performed according to the method of top3 previously described (Silva et al., 2006). The results at protein and peptide level were exported in *.csv files for subsequent analysis.