Computational protocol: Identification of Protein Partners in Mycobacteria Using a Single Step Affinity Purification Method

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[…] The protein pellets were dissolved in 50 μl of 100 mM NH4HCO3 and subjected to a standard procedure of trypsin digestion: the proteins were reduced with 10 mM DTT for 30 min at 56°C, alkylated with 55 mM iodoacetamide in darkness for 45 min at RT and digested overnight with 10 ng/μl trypsin. The resulting peptide mixtures were applied to RP-18 pre-columns of an HPLC system (Waters) using water containing 0.1% trifluoroacetic acid as the mobile phase, and transferred to a nano-HPLC RP-18 column (internal diameter: 75 μM, Waters) using an acetonitrile gradient (0%–35% ACN in 160 min) in the presence of 0.1% trifluoroacetic acid at a flow rate of 250 nl/min. The column outlet was directly coupled to the ion source of an Orbitrap Velos mass spectrometer (Thermo Scientific). A blank run ensured absence of cross-contamination from preceding samples.The mass spectrometer was operated in a data-dependent mode to automatically switch between Orbitrap MS and LTQ–MS/MS acquisition. Survey full-scan MS spectra (from m/z 300 to 2000) were acquired in the Orbitrap with a resolution of R  =  15,000 at m/z 400 (after accumulation to a target of 1,000,000 charges in the LTQ). The method used allowed sequential isolation of the most intense ions (up to 5, depending on the signal intensity) for fragmentation on the linear ion trap using collision-induced dissociation at a target value of 30,000 charges. The target ions selected for MS/MS were dynamically excluded for 60 s. Chromatographic peak apex detection triggered data dependent scans (expected peak width: 5 s, minimal signal threshold: 10,000 counts) with phase method activated and triggering window set to 30%. General MS conditions were as follows: electrospray voltage, 1.8 kV; no sheath and auxiliary gas flow. The ion selection threshold was 10,000 counts for MS/MS, and an activation Q-value of 0.22 and activation time of 30 ms were also applied.The raw files were processed, including peak list generation, using the MaxQuant (v1.3.0.5) computational proteomics platform and default parameters were used. The fragmentation spectra were searched using Andromeda search engine integrated into the MaxQuant platform against an M. smegmatis mc2155 protein database available at the CMR website (, 6878 entries, v15.1, Oct 15, 2004) or against an M. bovis BCG database (, NC_008769, 3952 entries). The databases were modified in-house to contain randomized sequences of all entries to control for false-positive identifications during analysis using the Andromeda search engine. The error ranges for the first and main searches were 20 ppm and 6 ppm, respectively, with 2 missed cleavages. Carbamidomethylation of cysteines was set as a fixed modification, and oxidation and protein N-terminal acetylation were selected as variable modifications for database searching. The minimum peptide length was set at 7 aa. Both peptide and protein identifications were filtered at a 1% false discovery rate and were thus not dependent on the peptide score. Enzyme specificity was set to trypsin, allowing cleavage of N-terminal proline. A ‘common contaminants’ database (incorporated in MaxQuant software) containing commonly occurring contaminations (keratins, trypsin etc.) was employed during MS runs.Bioinformatics analysis was performed using the Perseus tool (v1.3.0.4, Cox J., Max Planck, 2012). Contaminants and random protein identification were excluded. Proteins identified by less than 2 peptides were excluded from the results, except SH, the quantification peptide. Peptide and protein identification details, including scores, are provided in and . [...] For protein complex cross-linking, we selected the DNA-directed RNA polymerase, where the alpha subunit (rpoA, MSMEG_1524) was fused with the C-terminal GFP tag. The purification procedure was as described above, with the TEV cleavage buffer changed to a 10 mM HEPES (pH 8.0)-based buffer. The purified protein complexes eluted from the column were subjected to cross-linking. We used bis(sulfosuccinimidyl) suberate (BS3) as the cross-linker (Thermo Scientific), with an 8-carbon spacer arm (11.4 Å), according to the manufacturer’s protocol. Heavy (d4) and light (d0) versions of BS3 reagent were dissolved in DMSO and mixed at a 1∶1 ratio immediately before use. The d0/d4 mixture was used to induce stable and selective chemical cross-links between lysine (K) residues available on surfaces of purified proteins to fix potential interactions between protein partners. Next, 50 mM of the BS3 (d0/d4) mixture was added at a final concentration of 2 mM to purified proteins and incubated for 15 min at 4°C. The reaction was terminated by adding 10 μl of 3 M Tris solution (pH 8.0). The samples were precipitated with PRM, as described above. Subsequently, the proteins were digested overnight with 10 ng/ml trypsin (Promega) in 100 mM ammonium bicarbonate buffer at 37°C. The peptides were reduced in 10 mM DTT for 30 min at RT and alkylated in 55 mM iodoacetamide for 20 min at RT. Finally, trifluoroacetic acid was added at a final concentration of 0.1%.To determine protein compositions of the cross-linked samples, we used MaxQuant software (as described above). To search for the cross-linked peptides, we used pLink (pFind Studio) . The following parameters were used: precursor mass tolerance, 50 ppm; fragment mass tolerance, 20 ppm; cross-linker, light [d0]-BS3 and heavy [d4]-BS3 (cross-linking sites, K and protein N-terminus; xlink mass-shift, 138.0680796 and monolink mass-shift, 156.0786442); isotope shift, 4.0247 Da; fixed modification, C 57.02146 and enzyme, trypsin.We used. mgf files (Mascot Generic Files generated from. raw files by Mascot Distiller) and a protein database containing proteins found in a preceding MaxQuant search. All looplinks and monolinks were excluded from our obtained results. Only inter- or intra-molecular cross-links were used for further analysis. Molecular graphics and analyses were performed with the UCSF Chimera package from the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco (supported by NIGMS 9P41GM103311). […]

Pipeline specifications

Software tools MaxQuant, Perseus, pLink, pFind, Mascot Distiller, UCSF Chimera
Application MS-based untargeted proteomics
Diseases Tuberculosis