Speedvac centrifuge

The serum samples were firstly processed with ProteoExtract Albumin/IgG Removal Kit (Merck, Germany) to remove high abundant albumin and immunolgobulin G as the manufacturer’s instructions. Then the serum pools were ultrafiltered through Millipore 10 KD filters (Merck Millipore, Germany). The peptides were reduced using 5 mM dithiothreitol at 37°C for 45 min and alkylated using 10 mM iodoacetamide at room temperature keep away from light for 30 min. All the peptide mixtures were concentrated using a Speed-vac centrifuge (Eppendorf, Germany), dissolved in 0.1% formic acid, and analyzed in an ultra-performance liquid chromatograph (UPLC) system (Waters, Milford, MA) coupled with an LTQ Orbitrap Velos mass spectrometer (ThermoFisher Scientific, Waltham, MA) as previously reported.26 (link),27 (link) Proteome Discoverer software (version 2.4.0.305, Thermo Fisher Scientific, Waltham, MA) were used to process the raw data with search algorithms SEQUEST (version 1.3, Thermo Fisher Scientific, Waltham, MA) against Mycobacterium tuberculosis (strain ATCC 25618/H37Rv) database from Swiss-Prot database (release 2021_04). All the serum samples from the same condition were pooled and the LC-MS/MS analysis was performed in three technical replicates.

Dry protein was dissolved in 40 μl of SDC reduction and alkylation buffer (PreOmics GmbH, Martinsried, Germany) and boiled for 10 min at 95 °C while vortexing at 1200 rpm to denature the protein [7 (link)]. The lysate was digested for 5 h with LysC and trypsin (0.25 μg each) at 37 °C and 1200 rpm. Peptides were acidified to a final concentration of 0.1% trifluoroacetic acid (TFA) to quench the digestion reaction. Peptide concentration was estimated using Nanodrop and sample was loaded on two 14-gauge Stage-Tip plugs. Peptides were washed first with isopropanol/1% TFA (200 μl) and then 0.2% TFA (200 μl) using a centrifuge at 2000xg. Peptides were eluted with 60 μl of elution buffer (80% acetonitrile/1% ammonia) and dried at 60 °C using a SpeedVac centrifuge (Eppendorf, Concentrator plus). Dried peptides were redissolved and sonicated in 20 μl of 5% acetonitrile/0.1% TFA and concentration measured using the Nanodrop.

In-solution digestion was performed using in-solution tryptic digestion and guanidination kit (Cat #89895, Thermo Fisher Scientific, MA) following the instructions provided by the manufacturer. The protocol can be summarized as follow: 10 μg protein sample was added to 15 μL 50 mM ammonium bicarbonate containing 100 mM DTT solution. The volume was completed to 27 μL and incubated at 95°C for 5 minutes. Iodoacetamide (IAA) was added to the heated sample to a 10 mM final concentration and incubated in the dark for 20 minutes. Then, 1 μL of 100 ng/μl trypsin solution was added and incubated for 3 hours at 37°C. Another 1 μL of 100 ng/μL trypsin was added to the peptide mixture and incubated overnight at 30°C. After incubation, the solution was concentrated with a SpeedVac centrifuge (Eppendorf, CT) to dryness and the peptides were resuspended in 0.1% Formic Acid solution (FA) for the nLC-MS/MS analysis.