2.4 Mass spectrometry analysis (nLC-ESI-MS/MS)
B. canis RM6/66 (ATCC 23365) proteins were separated using a NuPage 4-12% Bis-Tris pre-cast gel (Life Technologies) at 200 V. Then proteins were stained with SimplyBlue SafeStain (Life Technologies). Stained gel was stored in 0.5% acetic acid at 4°C until protein analysis. Based on pattern profile identified by western blotting, two slices in the range 7-30 kDa were excised from the gel and the proteins were identified by nLC-MS/MS (Orbitrap QExactive-HF, Thermo Fisher) as previously reported [33-35].
Briefly, reduction with 10 mM DTT, alkylation with 55 mM IAA and trypsin digestion overnight at 37 oC were carried out as previously reported [36]. Five µl of peptides were injected on an UPLC EASY-nLC 1000 (Thermo Scientific) and separated on a homemade fused silica capillary column (75 μm i.d., length 25 cm), packed in house with ReproSil-Pur C18-AQ 1.9 µm beads (Dr. Maisch, Ammerbuch-Entringen, Germany). A gradient of eluents A (2% acetonitrile, 0.1% formic acid) and B (80% acetonitrile with 0.1% formic acid) was used to achieve separation, from 5% to 100% B (in 30 min, 250 nL/min flow rate). The nLC system was connected to a quadrupole Orbitrap QExactive-HF mass spectrometer (Thermo Fisher) equipped with a nano-electrospray ion source (Proxeon Biosystems). Top 15 method was applied. Raw data were processed with Proteome Discoverer (version 1.4.1.14, Thermo Scientific) and Mascot (version 2.6.0, Matrix Science) searching against B. canis, assuming a fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 10 ppm; specified enzyme was trypsin; carbamidomethylation of cysteine was set as a fixed modification; oxidation of methionine and acetylation of the N-terminus of proteins were set as variable modifications. Scaffold (version 4.8.9, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Only proteins with greater than 99.0% probability and containing at least 3 peptides (greater than 95% probability) were accepted. Therefore, only proteins detected in at least 2 out of 3 biological replicates were included in bioinformatics analysis.