Spectrum mill software

N-terminally his-MBP-tagged kinase domains of the nine canonical serine-threonine protein kinases from Mtb were expressed and purified from E. coli using a nickel column and then an S75 size exclusion column, in kinase buffer (50 mM Tris pH7.5, 150 mM NaCl, 20% glycerol). His-MBP-PonA1 (cytoplasmic domain) was expressed and purified from E. coli using Ni-NTA beads (Novagen) according to the protocol recommended by the manufacturer. Protein levels were measured by A280, and kinase reactions were started by mixing 1 μg of kinase, 10 μg of his-MBP-PonA1(cyto), 1mM MnCl₂ and 1mM ATP. Reactions were incubated at room temperature for 1 hour and stopped by addition of Laemmli buffer. Samples were heated, separated by SDS-PAGE, and detected by western blot using α-phospho-threonine antibody (Cell Signaling Technology). Kinase reactions for mass spectrometry were performed the same way, using only his-MBP-PknB and his-MBP-PonA1(cyto). The his-MBP-PonA1 protein band was cut out of the polyacrylamide gel and in-gel trypsin digested. Samples were analyzed by liquid chromatography (LC)/MS/MS with an Agilent 6520 Accurate-Mass Quadrupole Time-of-Flight instrument. Peptides were separated on a POROSHELL 300SB-C18 (2.1 × 75 mm, 5 μm) at a 0.5 mL/min flow rate, by using a linear gradient of increasing acetonitrile in water. Spectrum Mill software (Agilent) was used to identify peptides.

In-gel digestion with trypsin was carried out using the In-Gel Tryptic Digestion Kit (Thermo-Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, bands were destained in 200 mM ammonium bicarbonate, washed with 50:50 acetonitrile:water, reduced with 10 mM TCEP in 50 mM ammonium bicarbonate (pH 8.6) buffer for 10 minutes at 60 °C, and alkylated in 55 mM iodocetamide in 100 mM ammonium bicarbonate for one hour. Trypsin in ammonium bicarbonate at 20 ng/µL was added to cover gel pieces and incubated overnight at 30 °C. The digests were analyzed via LC-MSMS, using an Agilent 1200 LC system, an Agilent Chip-cube interface and an Agilent 6520 Q-TOF tandem mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Data files were transferred to an Agilent workstation equipped with Spectrum Mill software (Agilent Technologies, Santa Clara, CA, USA). The raw MS/MS data files were extracted, sequenced, and searched against the seven amino acid sequences shown in Figure 1. The LC-MSMS data is shown in Figure S1.

Trypsin digested proteins were identified by LC-ESI-QTOF as per the standard process described elsewhere (Kaplan et al., 2004 (link); Brun et al., 2020 (link)). Briefly, trypsin shaved proteins obtained from cell surface or ECM were treated with DTT and iodoacetamide for 1 h and the samples were digested with trypsin (1 μg/ml) for another 12–16 h at 37°C. The trypsin enzyme was inactivated by adding 5% Tri-Fluro acetic acid (TFA) to the peptide mixtures in tube. Peptide mixtures were pooled from three independent replicates, quantified and purified with C18 matrix column as per recommended protocol (Agilent, United States). Purified peptide mixtures were concentrated using a speed vacuum centrifugation unit at 4°C. Concentrated peptides were resuspended in 1% TFA and 5% acetonitrile (ACN) solution and submitted for identification by LC-ESI-QTOF. The MS/MS data was identified against S. aureus protein database (NCBI) using Spectrum Mill software (Agilent, United States) and spectral counts were used to calculate the fold change as described elsewhere (Liu et al., 2004 (link)).