ALA induced proteome of Mycobacterium smegmatis membrane fraction

ALA induced proteome of Mycobacterium smegmatis membrane fraction Shleeva Margarita1, Shashin Denis1, Demina Galina1, Smirnov Igor2 1A.N. Bach Institute of Biochemistry, Federal Research Centre ‘Fundamentals of Biotechnology’ of the Russian Academy of Sciences, Moscow, Russia 2Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency Medicine, Moscow, Russia. Description: A proteomic analysis was performed on the membrane proteins of M. smegmatis grown for 3 days on 7H9 medium supplemented with 3 mM ALA, under conditions where active secretion of porphyrins into the surrounding environment was observed. The bacterial lysate was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was separated into membrane and cytosolic fractions using ultracentrifugation at 100,000 × g for 2 h. The membrane fraction was washed with HEPES buffer three times using ultracentrifugation. Trypsin digestion: The membrane pellet was incubated at 4°C for 1 h with 10 μL of 10% sodium deoxycholate (DCNa) and 0.5 μL nuclease mix (GE Healthcare) and then was resuspended in 100 µL of 100 mM Tris-HCl buffer (pH 8.5) with 0.1% DCNa and protease inhibitor mix (Promega), following sonication at 4°C (4 times by 30 seconds). After centrifugation (14,000 g at 4°C) supernatant was collected and proteins were precipitated using chloroform-methanol. The pellet was resuspended in 100 µL 50 mM Tris-HCl buffer (pH 8.5) and protein concentration was measured using a BCA Assay Kit (Sigma). Disulfide bonds were reduced and alkylated by subsequent addition of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma) to a final concentration of 5 mM and chloroacetamide (Sigma) to a final concentration of 30 mM following incubation of resulting mixture at 80°C for 10 min. In the next step the Trypsin (Trypsin Gold, mass spectrometry grade, Promega) was added to the sample at a trypsin:protein ratio of 1:100 (w/w), following by incubation at 37°C for 2 h, after that the same amount of Trypsin was added again and incubation continued for another 16 h. Reaction was stopped by addition of trifluoroacetic acid (Sigma) to a final concentration of 0.5% (v/v). Peptide concentration was measured using a BCA Assay Kit (Sigma). The resulting peptide mixture was dried in a SpeedVac (Labconco) and prior to injection in to LC-MS it was resuspended in a 3% ACN solution containing 0.1% TFA. LC-MS analysis The LC-MS analysis of proteome samples was performed using Orbitrap Q Exactive HF-X (Thermo Fisher Scientific, Waltham, MA) mass spectrometer. For ionization a nano-electrospray (nano-ESI) source was used in conjunction with high-pressure nanoflow chromatograph UPLC Ultimate 3000 (Thermo Fisher Scientific, Waltham, MA). The lab-made reverse-phase column (ID 100mm with length 500mm of fused silica TSP100375 (Molex, Lisle, IL) was packed with phase Kinetex C18, 2.4 μm (Phenomenex, Torrance, CA) using pressure injection cell (Next Advance, Troy, NY). During HPLC run it was thermostatically controlled at 60°C. Samples were loaded in buffer A (0.1% Formic acid) and eluted with a linear (160 min) gradient of 3 to 55% buffer B (0.1% Formic acid, 80% Acetonitrile) at a flow rate 220 nl/min. Mass spectrometric data were stored during automatic switching between MS1 scans and up to 12 MS/MS scans (topN method). The target AGC value for MS1 scanning was set to 3x10e6 in the range 390-1400 m/z with maximum ion injection time of 50 ms and resolution of 60000. The precursor ions were isolated at a window width 2.0 m/z and then were fragmented by high-energy dissociation with a normalized collision energy of 30 eV. MS/MS scans were saved with a resolution of 30000 at 400 m/z and AGC of∙2e5 for target ions with a maximum ion injection time of 64 ms. The obtained data were processed using the Proteome Discoverer Ver 2.3 software package with standard settings (FDR for peptides 1%). The maximum allowable mass error was set at up to 10 ppm (MS mode) and 0.02 Da (MS/MS mode). Carbamidomethylation of cysteine was specified as a fixed modification, and methionine oxidation as a variable modification. The list of identified proteins comprises over 3100 entries. Proteins were identified based on one or more peptides, provided that each peptide was detected at least twice (this constitutes a PSM). The list is sorted in descending order of abundance in the samples compared to the control (column S in the table - log2(abundance), Sample vs Control). This study was funded by Russian Science Foundation grant 24-15-00221.