Dionex ultimate 3000 rslcnano liquid chromatography

Mtb-HAg digest peptide sequences were detected by LC–MS using a Q Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA) and Dionex Ultimate 3000 RSLCnano liquid chromatography (Thermo Fisher Scientific, MA, USA). The injection volume is 6 μL in LC–MS analysis. Column information: 300 µm idx 5 mm, Acclaim PepMap RSLC C18, 5 µm, 100 A (Thermo, 160,454); Acclaim PepMap 75 µm × 150 mm, C18, 3 µm, 100 A (Thermo Fisher Scientific, MA, USA). The elution conditions for LC were as follows: eluent A consisted of 0.1% formic acid in Milli-Q water, eluent B consisted of 0.1% formic acid and 80% acetonitrile in Milli-Q water, and the flow rate was set to 300 nL/min. The elution procedure was as follows: 0–5 min, 5% B; 5–45 min, 50% B; 45–55 min, 90% B; 55–65 min, 5% B. The MS parameters for Mtb-HAg analysis were set as follows: resolution = 70 k; scan range (m/z) = 350–1800; maximum injection time = 40 ms; AGC target = 300, 0000. The MS/MS parameters for Mtb-HAg analysis were set as follows: resolution = 17.5 k; AGC target = 100,000; maximum injection time = 60 ms; NCE/stepped NCE = 27. The mass spectrometry raw files were processed and converted by Proteome Discoverer 1.4 software to obtain MGF format files, and then the uniprot database (Mtb H37Ra (https://www.uniprot.org/taxonomy/419947) was searched using MASCOT (http://www.matrixscience.com/).

The proteomic analysis was carried out at Monash Biomedical Proteomics Facility, Monash, Australia. Data-dependent acquisition was performed on a QExactive™ Plus 1 mass spectrometer (Thermo Scientific, Waltham, MA, USA) coupled to a Dionex UltiMate^® 3000 RSLC nano Liquid Chromatography (LC) system (Thermo Scientific, Waltham, MA, USA) equipped with Acclaim PepMap RSLC analytical column (75 µm × 50 cm, nanoViper, C18, 2 µm, 100Å, Thermo Scientific, Waltham, MA, USA) and Acclaim PepMap 100 trap column (100 µm × 2 cm, nanoViper, C18, 5 µm, 100Å, Thermo Scientific, Waltham, MA, USA).