Mascot server version 2

Dried peptides were concentrated and de-salted using Zip-Tips C18 (microbed) (Millipore, Billerica, MA) according to the manufacturer’s instructions.
After elution from the Zip-Tip 0.5 µL of the de-salted peptides were spotted with α-cyano-4-hydroxycinnamic acid onto a ground steel MALDI target plate (Bruker Daltonics, Bremen, Germany). MALDI-TOF/TOF mass spectrometry (Ultraflex II, Bruker Daltonics, Bremen, Germany) was used for spectra acquisition in MS and MS/MS modes. Spectra processing and peak annotation were carried out using FlexAnalysis 3.0 and Biotools 3.2 (both Bruker Daltonics, Bremen, Germany).
Processed spectra were searched via an in-house Mascot server version 2.4.1 (Matrix Science, Boston, MA) and the software ProteinScape 2.1 (Bruker Daltonics, Bremen, Germany) in the UniProt database of sus scrofa using the following search parameters: global modification carbamidomethylation on cysteine; variable modifications oxidation on methionine; deamidation on asparagine and glutamine as well as formation of pyroglutamic acid; enzyme specificity trypsin; charge state z = 1; MS tolerance 100 ppm; MS/MS tolerance 1 Da; two missed cleavages allowed; significance threshold p < 0.05.

The in-gel tryptic digested peptides were injected into a nanoLC system (DIONEX, Ultimate 3000, UHPLC, RSLCnano, Thermo Scientific, Sunnyvale, CA, USA) and separated by a reversed-phase C18 PepMap column (75 µm × 15 cm; diameter, 2 µm; pore size, 120 Å) (Acclaim PepMap, Thermo Scientific, Sunnyvale, CA, USA). The nanoLC system was equipped with an electrospray quadrupole time-of-flight mass spectrometer (micrOTOF-Q II, Bruker Daltonics, Bremen, Germany). Peptides were eluted from the reversed-phase C18 column into the mass spectrometer using a linear gradient of 5–55% mobile phase B (0.1% formic acid in 80% acetonitrile) for 30 min at a flow rate of 0.3 µL/min. The data acquired by tandem mass spectrometry were processed using a data analysis software package (Bruker Daltonics GmbH). The processed data were used to identify proteins in a public database using the MASCOT server version 2.3 (Matrix Science, London, UK) by searching against the L. interrogans sequence database. The search parameters of MASCOT were set as follows: peptide mass tolerance, ±1.2 Da; MS/MS tolerance, ±0.6 Da; and variable modifications were set at carbamidomethylated cysteine and oxidized methionine residues.