Tof tof optics

Prior to mass spectrometry analysis, the 2,5-dihydroxybenzoic acid (DHB) matrix was added to a final concentration of 10 mg/mL in chloroform/methanol at a ratio of 90:10 v/v. 0.4 μL of cell solution and 0.8 μL of the matrix solution were deposited on the MALDI Target, mixed with a micropipette and left to dry. MALDI-TOF analysis was performed on a 4800 Proteomics Analyzer (with TOF-TOF Optics, Applied Biosystems) using the reflectron mode. Samples were analyzed operating at 20 kV in negative and positive ion mode, and three independent experiments were performed. Mass spectrometry data were analyzed using both Data Explorer version 4.9 from Applied Biosystems and R [9 ].
The MS data for each sample consists of around 130,000 pairs of (m/z, I) values, where m/z stands for the mass to charge ratio and I stands for the intensity value. m/z values range from around 399 to 4012 (Fig 1). Since there is variation in m/z values across samples and experiments, we cannot directly summarize the MS data into a M×N feature matrix of M features (peak positions or m/z values) and N samples and several steps of pre-processing is required as explained in “Data pre-processing”.

Cells were treated with BDQ during 18 hr and them lysed in water during 10 min at 37°C. Samples were heated at 90°C during 40 min in order to inactivate MTB, and were then washed three times to remove salts and contaminants that could preclude the analysis. Prior to mass spectrometry analysis, the 2,5-dihydroxybenzoic acid (Sigma-Aldrich, Saint-Louis, Missouri) matrix was added at a final concentration of 10 mg/mL in a chloroform/methanol mixture at a 90:10 (v/v) ratio; 0.4 μL of a cell solution at a concentration of 2 × 10⁵ to 2 × 10⁶ cells/mL, corresponding to ∼100–1,000 cells per well of the MALDI target plate (384 Opti-TOF 123 mm ×84 mm, AB Sciex), and 0.6 μL of the matrix solution were deposited on the MALDI target plate, mixed with a micropipette, and left to dry gently. MALDI-TOF MS analysis was performed on a 4800 Proteomics Analyzer (with TOF-TOF Optics, Applied Biosystems, Foster City, California) using the reflectron mode. Samples were analyzed operating at 20 kV in the negative and positive ion mode. Mass spectrometry data were analyzed using Data Explorer version 4.9 from Applied Biosystems.