Liquid chromatography tandem mass spectrometry (LC-MS/MS) for peptide analysis

According to the results of 2-DE analysis, we selected 30 spots from each image of the B. abortus mutants that had higher than 2-fold changes (increase or decrease) from those of B. abortus wild-type. LC-MS/MS for peptide analysis was performed on the selected spots.

Nano LC-MS/MS analysis was performed with an Easy n-LC chromatograph (Thermo Fisher, USA) and a LTQ Orbitrap XL mass spectrometer (Thermo Fisher) equipped with a nanoelectrospray source. Protein samples were separated on a C18 nano bore column (150 mm × 0.1 mm, 3 µm pore size; Agilent, USA). Mobile phase A for LC separation was 0.1% formic acid, 3% acetonitrile in deionized water, while mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was designed for a linear increase from 5% B to 30% B in 23 min, 30% B to 60% B in 3 min, 95% B in 3 min, and 3% B in 6 min. The flow rate was maintained at 1,500 nL/min. Mass spectra were acquired by using data-dependent acquisition with a full mass scan (350–1,200 m/z) followed by 10 MS/MS scans. For MS 1 full scans, the orbitrap resolution was 15,000 and the AGC was 2 × 10⁵. For MS/MS in the LTQ, the AGC was 1 × 10⁴ [15].