Data analysis software version 3

To identify target proteins, peptide mixtures were analysed by MALDI-FTICR-MS in a Bruker Apex Ultra, Apollo II combi-source (Bruker Daltonics, Bremen, Germany), with a 7 Tesla magnet (Magnex Corporation, Oxford, UK) as previously described [18,19]. Briefly, samples were desalted and concentrated using reverse phase Poros R2 (Applied Biosystems) and eluted directly to the MALDI target AnchorChip (BrukerDaltonics, Bremen, Germany) with the appropriated matrix, according to the manufacturer’s instructions. Matrix solution of α-cyano-4-hydroxycinnamic acid (CHCA; Fluka) was prepared at a concentration of 10 μg/μL in 50% ACN with 0.1% TFA. Monoisotopic peptide masses were determined using the SNAP 2 algorithm in Data Analysis software version 3.4 (BrukerDaltonics). External calibration was performed using the BSA tryptic digest spectrum, processed and analysed with Biotools 3.1 (BrukerDaltonics, Bremen, Germany).

Peptides separations were performed on an Ultimate3000 HPLC (Thermo). Tryptic peptides were loaded onto a trap column (PepMap100 C18, 5 μm, 100 Å, 300 μm ID, 5 mm) using buffer A (flow: 20 μl/min) and separated using reverse-phase C18 analytical column (PepMap100 C18, 3 μm, 100 Å, 75 μm i.d., 15 cm) with a linear gradient of buffer B (95% acetonitrile, 4.9% H2O, 0.1% formic acid) ranging from 0 to 50% within 70 min (flow: 300 nl/min). Eluted peptides were analysed by nanoESI ion trap mass spectrometer (HCTultra, Bruker) set to isolate and fragment the top 5 most abundant precursors. Electrospray voltage was set to 2000V and analysis mass ranges were 250–1500 m/z for MS and 200–3000 m/z for MS/MS. DataAnalysis software version 3.4 from Bruker was used to generate mgf files from raw data, with a precursor intensity threshold of 3.10⁵. The mgf files were submitted to local Mascot server using both PlasmoDB v13 (14 jan 2015, 5542 entries) and human SwissProt (6 march 2013, 20329 entries) databases. Search parameters were the following: MS and MS/MS mass tolerance = 0.5 Da; missed cleavages = 1; fixed modification = Carbamidomethyl (C), variable modification = Oxidation (M): FDR < 1% (search against the reversed merged database).

An Ultimate^® 3000 Nano-LC system (Thermo Scientific) was used for peptide separation. A microTOF-Q II (Bruker, Germany) was used to analyze MS and MS/MS spectra at m/z 400–2,000 and m/z 50–1,500, respectively. The acquisition was controlled by HyStar™ version 3.2 (Bruker). DataAnalysis™ software version 3.4 (Bruker) was used to convert raw data format (.d) files to mascot generic files (.mgf), which were further searched by Mascot software (Matrix Science, USA). A SwissProt bacterial database was set for protein identification.