Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Summary

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Abstract

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.

We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms¹. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

Introduction

c-di-GMP is a key second messenger used by most bacteria to control various aspects of their growth and behavior. For instance, c-di-GMP regulates cell cycle progression, motility and the expression of exopolysaccharides and surface adhesins^2-4. Through the coordination of such processes c-di-GMP promotes biofilm formation, a process which is associated with chronic infections of a range of pathogenic bacteria⁵. c-di-GMP is synthetized by enzymes called diguanylate cyclases (DGCs) that harbor a catalytic GGDEF domain⁴. Some DGCs possess an inhibitory site that down regulates the cyclase activity upon c-di-GMP binding. The degradation of c-di-GMP is catalyzed by two distinct classes of phosphodiesterases (PDEs) harboring either a catalytic EAL or HD-GYP domain^6,7.

The majority of the known effector proteins that directly bind c-di-GMP belong to one of only three classes of proteins: catalytically inactive GGDEF or EAL domains and PilZ domains, small molecular switches that undergo conformational changes upon c-di-GMP binding⁸. DGCs, PDEs and PilZ proteins are well characterized and their domains can be predicted in silico relatively safely. A particular interest is now focused on the identification of new classes of c-di-GMP effectors. Some c-di-GMP effectors with different binding motifs were described recently such as the CRP/FNR protein family Bcam1349 in Burkholderia cenocepacia or the transcriptional regulator FleQ in P. aeruginosa^9,10. In addition, c-di-GMP-specific riboswitches were recently identified and shown to control gene expression in a c-di-GMP-dependent manner¹¹. The c-di-GMP binding motifs of different effectors are only poorly conserved making bioinformatic predictions of such proteins difficult. To address this issue, we developed a biochemical method, which is based on the use of a c-di-GMP specific Capture Compound combined with mass spectrometry ^1,12,13.

We have recently engineered a novel trivalent c-di-GMP Capture Compound (cdG-CC, Figure 1)¹. This chemical scaffold is composed of: 1) a c-di-GMP moiety used as bait to capture c-di-GMP binding proteins, 2) a UV-photoactivatable reactive group used to cross link the cdG-CC to the bound proteins and 3) a biotin to isolate the captured proteins using streptavidin-coated magnetic beads. The cdG-CC can be used to directly and specifically capture c-di-GMP effectors from complex mixture of macromolecules as cell lysates. Capture Compound based and chemical proteomics based approaches have previously been reported to be applicable to a wide range of organisms, e.g. Caulobacter crescentus, Salmonella enterica serovar typhimurium and P. aeruginosa^1,14.

In this methodological paper, we provide an in depth description of the CCMS procedure using extracts of P. aeruginosa as an example. This study establishes CCMS as a powerful and versatile tool to biochemically identify novel components involved in small molecule signaling.

Protocol

1. Lysate Preparation

1. Grow P. aeruginosa cells in LB to the desired OD.
   NOTE: For guidance: use ≈ 100 ml culture/sample for stationary phase cultures and ≈ 500 ml cultur /sample for log phase cultures (OD_600nm = 0.5).
2. Pellet by centrifugation for 20 min at 5,000 x g.
3. Resuspend 0.5-1 g of pellet in 1 ml lysis buffer (6.7 mM MES, 6.7 mM HEPES, 200 mM NaCl, 6.7 mM KAc, DDT 1 mM, pH 7.5) and add protease inhibitor (complete mini, EDTA-free) as well as DNaseI.
4. Lyse the cells by 3 passages through a French pressure cell, at 20,000 psi (see Materials List).
5. Ultra-centrifuge the cell lysate at 100,000 x g for 1 hr at 4 °C.
6. Save the supernatant (go to step 2).
7. Wash the pellet with 1 ml 1x lysis buffer by pipetting up and down.
8. Ultracentrifuge at 100,000 x g for 1 hr at 4 °C.
9. Flash-freeze the pellet in liquid nitrogen and store at -20 °C until used for the capture of membrane proteins (see step 3).

2. Removal of Free c-di-GMP and Other Nucleotides (Soluble Fraction Only)

1. Wash a PD10 desalting column (see Materials List) with 10 ml of cold lysis buffer.
2. Pour the supernatant (≈ 1 ml) onto the PD10 in order to remove nucleotides.
3. Elute with 4 ml cold lysis buffer (500 µl steps).
4. Select the most concentrated fractions as determined by Bradford assay, and pool them.

3. Pellet Resuspension and Solubilization (Membrane Fraction Only)

1. Resuspend the pellet in 500 to 1,000 µl of 1x capture buffer (without detergent) (see Table 1).
   NOTE: The pellet is difficult to resuspend. It is advised to first pipette up and down with a pipette to roughly resuspend the pellet, then to use a syringe 27 G to well homogenize the solution.
2. Add 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM).
3. Incubate at 4 °C for at least 2 hr (or O/N) on a rotating wheel.
4. Ultracentrifuge at 100,000 x g for 1 hr at 4 °C.
5. Harvest the supernatant.

4. Protein Concentration Measurement

1. Measure the protein concentration by Bradford assay (by BCA assay for the membrane fraction).
2. Set the total protein concentration to 10 mg/ml.

5. Capture

1. Mix 300 µg of protein with 1 mM of GDP, GTP, ATP, CTP, and 20 µl of 5x capture buffer (100 mM HEPES, 250 mM KAc, 50 mM MgAc, 5% glycerol, pH 7.5) (Table 1). Adjust the reaction volume to 100 µl with H₂O.
   NOTE: the total volume includes the volume of the cdG-CC and c-di-GMP in case of the competition control (see step 5.3 and Table 2).
   NOTE: all experiments were performed in 200 µl 12-tube PCR strips (Thermo Scientific).
   NOTE: for the membrane fraction, make sure to always keep the DDM concentration above the critical micelle concentration (0.01% w/v) until the protein solubilization step in 8 M Urea (Step 7.1).
2. Incubate at 4 °C for 30 min on a rotating wheel.
3. Add 10 µM cdG-CC (final concentration).
   NOTE: include a control without cdG-CC (referred as “bead control”), and a control supplemented with 1 mM c-di-GMP (“competition control”) (see Table 2).
   NOTE: the cdG-CC concentration can be adjusted from 1 to 10 µM.
4. Incubate at 4 °C for at least 2 hr (O/N for the membrane fraction) on a rotating wheel, in the dark.
5. After a short spin, cross-link by activation of the reactive moiety of the cdG-CC with UV light for 4 min, using a CaproBox (see Materials List, λ = 310 nm, Irradiance ≥10 mW/cm², distance from the source = 2 cm). NOTE: remove the lid of the strips prior cross-linking.
6. Add 25 µl 5x wash buffer (5 M NaCl, 250 mM Tris, pH 7.5) and 50 µl of well resuspended streptavidin magnetic beads, gently homogenize.
7. Incubate at 4 °C for 1 hr on a rotating wheel.

6. Washing Steps

NOTE: (Magnet: see Materials List). Start with a capture of the magnetic beads in the PCR strip lid, with the magnet. Then replace the PCR strip by a new one containing the next washing solution. Remove the magnet and resuspend the beads, and incubate 2 min. Spin down and replace the lid by a fresh lid.

1. Washing steps (soluble fraction only)
   1. Wash 6 times in 200 µl 1x washing buffer.
   2. Wash once in 200 µl HPLC grade H₂O.
   3. Wash 6 times in 200 µl 80% acetonitrile.
   4. Wash 2 times in 200 µl HPLC grade H₂O.
2. Washing steps (membrane fraction only)
   1. Wash 5 times in 200 µl 1x washing buffer + 0.1% DDM.
   2. Wash 2 times in 200 µl 1x washing buffer + 0.05% DDM.
   3. Wash once in 200 µl 1x washing buffer + 0.025% DDM.
   4. Wash once in 200 µl 1x washing buffer + 0.0125% DDM.
   5. Wash 3 times in 200 µl 100 mM ABC (ammonium bicarbonate, NH₄CO₃) + 2 M Urea.

7. MS Sample Preparation

1. Resuspend the beads (directly in the lid) in 20 µl 100 mM ABC (100 mM ABC + 8 M Urea for the membrane fraction) and transfer into 1.5 ml tubes.
2. Membrane fraction only: incubate at 60 °C for 5 min, shaking at 500 rpm.
3. Add 0.5 µl 200 mM TCEP (tris(2-carboxyethyl)phosphine) and incubate at 60 °C for 1 hr, shaking at 500 rpm. Cool down to 25 °C.
4. Add 0.5 µl of freshly prepared 400 mM iodoacetamide and incubate at 25 °C for 30 min, shaking at 500 rpm and in the dark.
5. Add 0.5 µl 0.5 M N-acetyl-cysteine and incubate at 25 °C for 10 min, shaking at 500 rpm.
6. Membrane fraction only: add 1 µl Lys-C and incubate at 37 °C, O/N.
7. Add 2 µg trypsin and incubate O/N at 37°C, shaking at 500 rpm (wrap in Parafilm to prevent drying).
   NOTE: the samples can be stored at -20 °C at this stage.
   NOTE: membrane fraction only: add 100 mM ABC to adjust the urea concentration to <2 M before addition of trypsin.
8. Briefly spin down the tubes and collect beads with the magnet.
9. Transfer the supernatant into a new 1.5 ml tube (repeat this step if beads are left).
10. Add 5 µl 5% TFA (trifluoroacetic acid) + 1 µl 2 M HCl (15 µl 5% TFA+ 5 µl 2 M HCl for the membrane fraction).
11. Condition C18 MicroSpin columns (The Nest Group, MA, USA) with 150 µl acetonitrile (spin 20 sec at 2,400 rpm).
12. Equilibrate the C18 columns 2 times with 150 µl 0.1% TFA (spin 20 sec, 2,400 rpm).
13. Load the sample and spin 2 min, 2,000 rpm.
14. Reload the flow-through onto the column and repeat the spinning step (2 min, 2,000 rpm).
15. Wash 3 times with 150 µl 0.1% TFA, 5% acetonitrile (20 sec, 2,400 rpm).
16. Take a new tube and elute twice with 150 µl 0.1% TFA, 50% acetonitrile (2 min, 2,000 rpm).
17. Dry the peptides in a speed-vac.
18. Resuspend in 40 µl 98% H₂O, 2% acetonitrile, 0.15% formic acid.
19. Sonicate 20 sec (pulse cycle 0.5, amplitude 100%; see Materials List) and spin down 5 sec, 12,000 rpm (benchtop centrifuge). Vortex 10 sec, and spin down 5 sec, 12,000 rpm. Transfer in a HPLC vial for LC-MS/MS analysis.
20. Freeze at -20 °C.
    NOTE: the samples can be stored at -20 °C at this stage.

8. LC-MS/MS Analysis

1. Run a nano-LC (nano-LC systems) equipped with a RP-HPLC column (75 μm x 37 cm) packed with C18 resin (Magic C18 AQ 3 μm) using a linear gradient from 95% solvent A (0.15% formic acid, 2% acetonitrile) and 5% solvent B (98% acetonitrile, 0.15% formic acid) to 35% solvent B over 60 min at a flow rate of 0.2 µl/min.
2. Analyze the peptides using LC-MS/MS (dual pressure LTQ-Orbitrap Velos mass spectrometer, connected to an electrospray ion source).
   NOTE: The data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 60,000 FWHM followed by MS/MS scans in the linear ion trap of the 20 most intense ions. To increase the efficiency of MS/MS attempts, the charged state screening modus was enabled to exclude unassigned and singly charges ions. Collusion induced dissociation was triggered when the precursor exceeded 100 ion counts. The dynamic exclusion duration was set to 30 sec. The ion accumulation time was set to 300 msec (MS) and 50 msec (MS/MS).

9. Database Search

1. Download the P. aeruginosa NCBI-database via the NCBI homepage (http://www.ncbi.nlm.nih.gov/).
2. Convert the MS raw spectra into mascot generic files (mgf) using the MassMatrix conversion tool (http://www.massmatrix.net/mm-cgi/downloads.py).
3. Search this mgf file using MASCOT version 2.3 against the P. aeruginosa NCBI-database containing forward and reverse-decoy protein entries.
4. Perform an in silico trypsin digestion after lysine and arginine (unless followed by proline) tolerating two missed cleavages in fully tryptic peptides.
5. Set the database search parameters to allow oxidized methionines (+15.99491 Da) as variable modifications and carboxyamidomethylation (+57.021464 Da) of cysteine residues as fixed modification. For MASCOT searches using high-resolution scans, set the precursor mass tolerance to 15 p.p.m. and set the fragment mass tolerance to 0.6 Da. Finally, set the protein FDR to 1%.
6. Import the Mascot searches of P. aeruginosa CCMS experiments into Scaffold (Proteomesoftware, Version 3), set the parameters to obtain a protein FDR close to 1%, and extract the total spectral counts.
7. For the representative results presented in this paper, we used a paired T test to compare the experiment with the competition control, and only considered hits with a p value below 0.1, and a spectral count ratio above 2 (experiment spectral counts/competition control spectral counts).
8. Data can be exported in any spreadsheet software for further analysis.

10. Label-free Quantification

1. Import the raw files into Progenesis LC-MS software (Nonlinear Dynamics, Version 4.0).
2. Perform LC-MS alignment and feature detection in default settings.
3. Export the data in mgf format from Progenesis LC-MS.
4. Search the MS/MS spectra using the MASCOT against the NCBI P. aeruginosa database containing forward and reverse-decoy protein entries.
5. Import the database search results into Progenesis LC-MS and map the peptides identifications to MS1 features.
6. For data evaluation (calculation of significance levels, fold-change ratios) the ProteinSQAnalysis script of SafeQuant was used (threshold: q value below 0.1, spectral count ratio above 2).

Results

To identify novel c-di-GMP effectors in P. aeruginosa we systematically used CCMS to analyze the soluble and membrane fractions of P. aeruginosa strain PAO1 from a log phase culture (OD₆₀₀ = 0.5). Here we summarize and discuss representative results of this fishing expedition. Four independent biological replicas were used. For each experiment two different cdG-CC concentrations were used (5 µM and 10 µM). To probe for specificity, experiments were carried out in the presence or absence of 1 ...

Discussion

Special care should be taken at several steps of the protocol. The protein concentration is a critical parameter with a concentration of 10 mg/ml being difficult to reach when cells are grown under specific growth conditions (e.g. biofilms or small colony variants). Thus, the pellet resuspension should be performed in a low volume of lysis buffer. Protein concentrations can be decreased to 8 mg/ml. Compared to the method published by Nesper et al.¹, we added various nucleotides to the capture...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
caproBox	caprotec bioanalytics	1-5010-001 (220 V)	UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation
caproMag	caprotec bioanalytics	included in the CCMS Starter Kit	For easy handling of magnetic particles without pipetting
c-di-GMP caproKit	caprotec bioanalytics	upon request	The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer
Disposable PD-10 Desalting Columns	GE Healthcare	17-0851-01
12-tube PCR strips	Thermo Scientific	AB-1114
UIS250v sonicator with VialTweeter	Hielscher ultrasound technology	UIS250v and VialTweeter
Miniature French Pressure Cell	Thermo Electron Corporation	FA-003