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.