Thermo quadrupole orbitrap q exactive mass spectrometer

Total protein concentration in AF samples was individually determined by a BCA assay (Micro BCA Protein Assay Kit, Thermo Fisher Scientific, Bremen, Germany). The pooled AF samples of SPTD case and TD control groups were generated by combining equal amounts of 10 individual AF samples from each group and further filtering them by centrifugation for 5 min at 16,000g and 4 °C. The 14 most abundant proteins were removed from 200 μg of pooled AF samples and subjected to tryptic digestion followed by high-pH reversed-phase fractionation as described in the Supplementary Material.
LC-MS/MS analyses of the fractionated peptide samples were performed using an online Thermo Easy nLC 1000 (Thermo Fisher Scientific, Bremen, Germany) system interfaced to a Thermo quadrupole-orbitrap Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany), controlled by Xcalibur version 2.0.6 software (Thermo Fisher Scientific, San Jose, CA, USA) as described in the Supplementary Material.

Quantification of total protein amount in each plasma sample was performed using a bicinchoninic acid assay (Micro BCA Protein Assay Kit, Thermo Fisher Scientific, Bremen, Germany). The pooled plasma samples from the TB control and SPTB case groups (10 samples per group; 4 mg per group) were generated by combining equal amounts (400 μg) of 10 individual plasma samples from each group and further filtered by centrifugation for 5 min at 16,000 × g and 4°C. Subsequently, the pooled plasma samples within each group (4 mg per group) were subjected to immunoaffinity depletion for removal of the top 14 high-abundance proteins, as described in the Supplementary Materials. The depleted plasma samples (300 μg per group) were digested by trypsin, followed by high-pH reversed-phase fractionation, as described in the Supplementary Materials.
The fractionated peptide samples were then analyzed in triplicate using an online Thermo Easy nLC 1000 system (Thermo Fisher Scientific, Bremen, Germany) interfaced with a Thermo quadrupole-orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), controlled by Xcalibur version 2.0.6 software (Thermo Fisher Scientific, San Jose, CA, USA), as described in the Supplementary Materials.