A study of Plasmodium UIS4 protein interactors
To identify interacting partners of the parasitophorous vacuole membrane-resident protein UIS4 (PbUIS4) we employed immunoprecipitation followed by mass spectrometry analysis. Anti-PbUIS4 antibody (SICGEN, Cat# AB0042-200) was used to probe the lysates of the hepatoma cell line Huh7, infected either with PbWT or Pbuis4- parasites. Immunoprecipitation was carried out at 24 h post-infection (h.p.i). The immunoprecipitated proteins were resolved by SDS-PAGE and visualized by silver staining (Figure pannel: MBana_Immunoprecipitation_SDS_SilverStaining.pdf). Three differential bands that were detected in the PbWT immunoprecipitate were excised and analyzed by mass spectrometry (Figure pannel – highlighted by the grey boxes). The regions corresponding to the molecular weights of those bands in the lane of immunoprecipitated proteins obtained from Pbuis4- infected cells were also excised and analyzed as controls (Figure pannel - highlighted by the green boxes). Excised protein bands were destained, reduced, alkylated, and digested with trypsin (Promega) overnight at 37⁰C (6.7 ng/μl). The tryptic peptides were actively extracted from the bands, desalted, and concentrated using POROS R2 (Applied Biosystems) and, eluted directly onto the MALDI plate using 0.6 μl of 2.5 mg/ml CHCA (alpha-cyano-4-hydroxycinnamic acid, Sigma) in 50 % (v/v) acetonitrile and 5 % (v/v) formic acid. Data acquisition was performed on positive reflector MS and MS/MS modes using a 4800plus MALDI-TOF/TOF (ABSciex) mass spectrometer and using 4000 Series Explorer Software v.3.5.3 (Applied Biosystems). External calibration was performed using CalMix5 (Protea). The twenty-five most intense precursor ions from the MS spectra were selected for MS/MS analysis. The raw MS and MS/MS data were analyzed using the Protein Pilot Software v. 4.5 (ABSciex) with the Mascot search engine (MOWSE algorithm). The search parameters were as follows: monoisotopic peptide mass values were considered, maximum precursor mass tolerance (MS) of 50 ppm and a maximum fragment mass tolerance (MS/MS) of 0.3 Da. The search was performed against the SwissProt protein database with no taxonomic restriction. A maximum of two missed cleavages was allowed. Carboxyamidomethylation of Cysteines, oxidation of methionine and N-Pyro Glu of the N-terminal Q were set as variable modifications. Protein identification was only accepted when significant protein homology scores were obtained (P<0.05, scores higher than 70) and at least one peptide was fragmented with a significant individual ion score (P<0.05). Mass Spectrometry Analysis of raw data with Mascot Search Results list for each excised sample are listed (Excel file: MBana_Mass Spectrometry Analysis_Raw Data).