A tailored phosphoaspartate probe unravels CprR as a response regulator in Pseudomonas aeruginosa interkingdom signaling
Allihn PWA, Hackl MW, Ludwig C, Hacker SM, Sieber SA. A tailored phosphoaspartate probe unravels CprR as a response regulator in Pseudomonas aeruginosa interkingdom signaling. Chem Sci [Internet]. 2021; Available from: http://dx.doi.org/10.1039/D0SC06226J
- Organism: Pseudomonas aeruginosa PAO1
- Instrument: Q Exactive Plus
Activity-based protein profiling, isotopically labeled tags, phosphorylation, response regulator, parallel reaction monitoring, adaptive resistance, antimicrobial peptide, P. aeruginosa
Lab head: Stephan Sieber
Submitter: Patrick Allihn
Pseudomonas aeruginosa is a difficult-to-treat Gram-negative bacterial pathogen causing life-threatening infections. Adaptive resistance (AR) to cationic peptide antibiotics such as polymyxin B impairs the therapeutic success. This self-protection is mediated by two component systems consisting of a membrane-bound histidine kinase and an intracellular response regulator (RR). As phosphorylation of the key RR aspartate residue is transient during signaling and hydrolytically unstable, the study of these systems is challenging. Therefore, we applied a tailored reverse polarity chemical proteomic strategy to capture this transient modification and read-out RR phosphorylation in complex proteomes using a nucleophilic probe. Analysis of Bacillus subtilis and P. aeruginosa proteomes revealed the detection of multiple phosphoaspartate sites, which closely resembled the conserved RR sequence motif. With this validated strategy we dissected the signaling of dynorphin A, a human peptide stress hormone, which is sensed by P. aeruginosa to prepare AR. Intriguingly, our methodology combined with parallel reaction monitoring identified CprR as an unprecedented RR in dynorphin A interkingdom signaling.
Using our probe (HA-yne) together with our isotopically labeled desthiobiotin azide (isoDTB) tags, we applied a tailored reverse polarity activity-based protein profiling (RP-ABPP) strategy to examine aspartate phosphorylated sites in P. aeruginosa upon dynorphin A treatment.
PRM method development: Based on the results of the quantitative data dependent acquisition (DDA) isoDTB experiments, the most interesting HA-yne modified peptides with the highest light to heavy MS1 ratios (L/H) were chosen for PRM measurements. The corresponding peptides from the response regulators CprR and ParR were selected for fragmentation, showing the highest or a so far uncharacterized MS1 ratio (light to heavy), respectively. Additionally, two peptides (from response regulators GacA and PhoP) with an MS1 ratio of roughly one, were chosen as controls. Precursors for fragmentation were selected based on their respective most intense charge state from the DDA measurements analyzed by MS1 Filtering using Skyline (version 188.8.131.526). Experimental spectral libraries were built within Skyline using DDA and PRM isoDTB data processed with MaxQuant. For retention time comparison, PROCAL retention time peptides (JPT Peptide Technologies) were used, consisting of 40 non-naturally occurring peptides. PROCAL peptides were spiked into the samples (final quantity: 100 fmol/peptide). For 34 PROCAL peptides only MS1 chromatogram information was acquired in PRM mode, while five PROCAL peptides were also selected for fragmentation.
isoDTB-ABPP Experiments in dynorphin A treated P. aeruginosa: P. aeruginosa overnight cultures were inoculated in fresh minimal medium and grown to OD600 = 0.8-1.0. Cells were harvested, washed with PBS, resuspended to OD600 = 1 in PBS and incubated at 37 °C. Dynorphin A (1-13) (final concentration: 10 µM) or DMSO were added and incubated for 1 min at 37 °C. The cells were harvested, washed with PBS and resuspended to OD600 = 40 in 1 mL of HA-yne buffer (20 mM HEPES, pH 4.0, 125 mM HA-yne, 1% (w/v) LDAO). Cells were lyzed by sonication and the reaction proceeded for 1 h at 37 °C. Fractions were separated by centrifugation and the insoluble fraction was washed twice with PBS and stored at -20 °C until subjection to click chemistry. The soluble fraction was precipitated acetone (-80 °C) and incubated overnight at -20 °C. The precipitate was centrifuged and washed twice with MeOH (-80 °C. Soluble and insoluble fractions were resuspended in 0.8% SDS in PBS and protein concentration of both fractions was determined by BCA assay and adjusted to 0.5 mg/mL with 0.8% SDS in PBS. Dynorphin A treated samples were clicked to the light isoDTB tag and DMSO treated samples were clicked to the heavy isoDTB tag by addition of TBTA ligand, the respective isoDTB tag, TCEP and CuSO4. The click reaction was incubated for 1 h at room temperature and quenched by combination of light and heavy isoDTB-tagged samples into acetone (-80 °C).
MS Sample Preparation for RP-ABPP and isoDTB-ABPP Experiments: Precipitates were centrifuged and washed twice with MeOH (-80 °C). Pellets were dissolved in 8 M urea in 0.1 M triethylammonium bicarbonate and after reduction of disulfides by addition of dithiothreitol (DTT) and incubation at 37 °C for 45 min, free thiols were alkylated by adding iodoacetamide (IAA) and incubation in the dark at 25 °C for 30 min. Remaining IAA was quenched by addition of DTT and incubation at 25 °C for 30 min. 0.1 M TEAB was added to obtain a urea concentration of 2 M for trypsin digestion. 20 µL of 0.5 µg/µL trypsin were added and samples were incubated at 37 °C overnight. This solution was added to washed streptavidin agarose beads in 0.2% nonyl phenoxypolyethoxylethanol (NP-40 alternative). The samples were incubated by rotation at room temperature for 1 h. To remove unbound peptides, the beads were centrifuged and the supernatant was removed. The beads were resuspended in 0.1% NP 40 alternative in PBS and transferred to a centrifuge column. The beads were washed with 0.1% NP 40 alternative, PBS and ddH2O. The peptides were eluted by addition of elution buffer (0.1% formic acid (FA) in 1:1 ACN/H2O) and two more elution steps, followed by centrifugation. The solvent was removed using a vacuum centrifuge and samples were dissolved by addition of 30 µL 1% FA in H2O and sonication for 3 min. The samples were filtered and transferred into MS vials for LC MS/MS analysis.
PRM LC-MS/MS Analysis: For PRM measurements, PROCAL retention time peptides were spiked into the samples (v/v 1:6) directly before measurement. 6 µL of sample were injected in order to obtain similar intensities as in previous DDA measurements and 100 fmol/peptide of the PROCAL retention time peptides. Samples (injection volume: 5 µL) were analyzed with a nano HPLC system coupled to a Q Exactive Plus mass spectrometer. Samples were loaded on a C18 trap column, washed with 0.1% TFA and separated on a C18 column with a flow of 300 nL/min using buffer A (0.1% FA in H2O) and buffer B (0.1% FA in ACN): 5% B for 7 min, 5 40% B in 105 min, 40 60% B in 10 min, 60 90% B in 10 min, 90% B for 10 min, 90 5% B in 0.1 min, 5% B for 9.9 min. For PRM measurements, the mass spectrometer was operated in PRM mode. Full MS (MS1) scans were acquired at a resolution of 70,000, a scan range of m/z = 300 1500 Th, an automatic gain control (AGC) target of 3e6, and a maximum injection time of 80 ms. Targeted MS2 scans were acquired at a resolution of 17,500, an AGC target of 1e5, and a maximum injection time of 100 ms. The number of targeted precursors was adjusted to maintain a maximum cycle time of 2 s for at least 8 points across the peak in a non-scheduled PRM measurement. In total, 4 different HA yne-modified peptides from proteins CprR, ParR, GacA and PhoP (light/heavy isoDTB version) and 5 PROCAL peptides were targeted (in comparison for DDA measurements: Top10). Quadrupole isolation of the precursor was set to a window of 1.6 Th. Fragment ions were generated using higher-energy dissociation (HCD) with a normalized collision energy (NCE) of 27% and detected in the orbitrap.
PRM Data Analysis: PRM data analysis was performed using the Skyline-daily (64 bit) software (version 184.108.40.2066). For all target peptides, the 6 most intense fragment ions (top6) were automatically picked by Skyline using the generated experimental spectral library. Raw PRM data were also processed by MaxQuant in order to visualize in Skyline the exact time point of successful peptide identification for any given MS2 spectrum. Peak picking, peak integration and transition interferences were reviewed and integration boundaries were adjusted manually in Skyline, if necessary. Mass accuracy information (“average mass error [ppm]”), correlation of fragment ion intensities between the detected light and heavy peptides (“dot product L/H”) and correlation of fragment ion intensities between the detected peptides measured by PRM and the experimental library spectrum from Skyline (“library dot product” separately for light and heavy) were exported from Skyline. Peptide identifications with a dot product L/H > 0.9 and a library dot product > 0.85 were included for the overall ratio (L/H) calculation. The ratio of the respective MS2 peak areas (“total area fragment” L/H) was used for the ratio (L/H) calculation.
Created on 11/11/20, 6:25 AM