Chemical proteomics for identifying short-chain fatty acid modified proteins in Salmonella

Abstract

Microbiota-metabolized small molecules play important roles to regulate host immunity and pathogen virulence. Specifically, microbiota generates millimolar concentration of short-chain fatty acid (SCFA) that can directly inhibit Salmonella virulence. Here, we describe chemical proteomic methods to identify SCFA-modified proteins in Salmonella using free fatty acids as well as their salicylic acid derivatives. In addition, we include CRISPR-Cas9 gene editing protocols for epitope-tagging of specific proteins to validate SCFA-modification in Salmonella. These protocols should facilitate the discovery and functional analysis of SCFA-modified proteins in Salmonella microbiology and pathogenesis.

1. Introduction

The microbiota and diet play important roles to modulate host immunity and control susceptibility to enteric pathogens (Belkaid and Hand, 2014; Buffie and Pamer, 2013). Increased microbial infections are observed when there are dysbiosis of gut microbiota or significant alterations in diet (Buffie and Pamer, 2013). Specifically, gut microbiota generate structurally diverse small molecules (Donia and Fischbach, 2015), which can modulate host immunity and directly regulate pathogen infection (Lee and Hase, 2014).

Salmonella enterica Typhimurium are Gram-negative enteric pathogens and are one of the leading causes of gastroenteritis (Ao et al., 2015). Humans are infected by Salmonella primarily through ingestion of contaminated food or water (Haraga et al., 2008). Salmonella can then survive the low pH and degradative environment of the stomach and enter the small intestine where the bacterium actively invades and crosses the physical barrier of the gut. Salmonella evolves two major Salmonella pathogenicity islands (SPI-1 and SPI-2) which encodes genes required for the early (invasion of non-phagocytic epithelial cells) and late (survival and replication in macrophages) stages of infection (Galán and Wolf-Watz, 2006; LaRock et al., 2015). The type III protein secretion system (T3SS) encoded by SPI-1 delivers effector proteins to host cells. Salmonella with deletion of T3SS components show significantly attenuated infection ability (Galán, 2001).

Short-chain fatty acids (SCFAs) are important gut microbiota-generated metabolites and their concentration in the mammalian gut ranges from 1–100 mM (Cummings et al., 1987). Among all the SCFAs, butyrate is reported to directly inhibit the expression of SPI-1 genes in vitro (Gantois et al., 2006). Moreover, recolonization with butyrate-producing Clostridia attenuates S. Typhimurium infection in vivo (Rivera-Chávez et al., 2016). To identify the protein targets of butyrate in S. Typhimurium, we employed a SCFA chemical reporter (Pentynoate, Alk-3) and label-free quantitative proteomics (Zhang et al., 2020a). We found site-specific acylation of Salmonella virulence regulator HilA attenuates Salmonella infection. Furthermore, inspired by mechanistic investigation of microbiota-derived short chain fatty acid (SCFA) acylation of bacterial virulence factors, we explored acylation reagent, acetylsalicylic acid derivatives, and discovered that SCFA analogs such as butyryl-salicylic acid showed significantly improved anti-infective activity against Salmonella Typhimurium (Yang et al., 2020).

The combination of specific metabolite reporters and chemical proteomics enables the investigation of metabolite-protein modifications and interactions (Grammel and Hang, 2013; Parker and Pratt, 2020; Zhang et al., 2020b). Herein, we describe a chemical proteomics method to identify short-chain fatty acid (SCFA)-modified proteins in Salmonella (Figure 1). Alk-3, the reporter for butyrate, modified many Salmonella proteins that can be imaged and enriched via Cu^I-catalyzed azide-alkyne cycloaddition (CuAAC) with azide-fluorophore or azide-biotin, respectively (Zhang et al., 2020a). In addition, we describe the labeling of Salmonella proteins with SCFA-salicylic acid derivatives, which more effectively inhibit bacterial virulence and pathogenesis in vivo (Yang et al., 2020). Alk-aspirin was firstly used to detect aspirin-dependent protein modification in mammalian cells (Bateman et al., 2013). We found alk-aspirin exhibited similar activity with butyryl-salicylic acid and was applied to explore SCFA-salicylic acid modified proteins in Salmonella. We also include CRISPR-Cas9 gene editing protocols for epitope-tagging of specific proteins to validate SCFA-modification in Salmonella (Fig. 2).

Figure 1.

Working flow of chemical proteomics for identifying short-chain fatty acid modified proteins in Salmonella, modified from ref. (Yang et al., 2020).

Figure 2.

Schematic of CRISPR-Cas9 genome editing in S. Typhimurium, modified from ref. (Zhang et al., 2020a).

2. In-gel profiling of SCFA-reporter labeled Salmonella total cell lysates

2.1. Equipment

37 °C incubator with shaker

Refrigerated centrifuge

Water bath sonicator

Microplate readers (BioTek)

Vortex mixer

−20 °C freezer

Block heater

Criterion vertical electrophoresis cell (Bio-Rad)

Typhoon 9400 imager (Amersham Biosciences)

ChemiDoc Imaging System (Bio-Rad)

2.2. Materials

Salmonella Typhimurium strain ATCC14028S

Miller LB Broth

Miller LB Broth with 300 mM NaCl (SPI-1 inducing LB)

Pentynoate (Alk-3)

PBS buffer

Nonidet P-40

EDTA-free protease inhibitor cocktail (Roche)

Lysozyme (Sigma)

Benzonase (Millipore)

BCA Protein Assay Kit (Thermo)

az-Rho (Rangan et al., 2010)

tris(2-carboxyethyl)phosphine hydrochloride (TCEP)

tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)

t-butanol

DMSO

CuSO₄

Methanol

1X Laemmli buffer

4–20% Tris-HCl gel (Bio-Rad)

Coomassie Blue Stains (Bio-Rad)

2.3. Protocol

2.3.1. Preparation of Salmonella total cell lysates

1. Salmonella Typhimurium strain 14028S WT was inoculated in 4 mL Miller LB. Bacteria was cultured overnight at 37 °C with 220 rpm shaking.

2. 1:50 dilutions of Salmonella Typhimurium strain 14028S WT overnight culture were grown in 4 mL SPI-1 inducing LB (with or without reporters) for 4 h at 37 °C with 220 rpm shaking.

3. S. Typhimurium cells were pelleted at 15000 g for 1 min, and pellets were washed with cold PBS once.

4. The pellet was lysed with 200 μL lysis buffer (phosphate-buffered saline (PBS) containing 0.5% Nonidet P-40, 1X EDTA-free protease inhibitor cocktail, 0.5 mg/mL lysozyme in dH₂O, and 1:1,000 dilution of Benzonase). After re-suspension, pellets were sonicated for 10 sec for 3 times, then were incubated on ice for 30 min. Cell lysates were centrifuged at 8200 g for 5 min to remove cell debris and supernatants were collected.

5. Protein concentration was estimated by BCA assay with BCA Protein Assay Kit.

2.3.2. In-gel fluorescence analysis of SCFA-reporter labeled Salmonella total cell lysates

1. From the alk-3-treated or control total cell lysates prepared as described above, 45 μL of each total cell lysates (~50 μg) was added with 5 μL of click chemistry reagents as a 10X master mix (az-Rho: 0.1 mM, 10 mM stock solution in DMSO; tris(2-carboxyethyl)phosphine hydrochloride (TCEP): 1 mM, 50 mM freshly prepared stock solution in dH₂O; tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA): (0.1 mM, 2 mM stock in 4:1 t-butanol: DMSO); CuSO₄ (1 mM, 50 mM freshly prepared stock in dH₂O).

2. Samples were mixed well and incubated at room temperature for 1 h.

3. After incubation, samples were mixed with 200 μL cold methanol and incubate at −20 °C overnight.

4. Sample proteins were precipitated at 18000 g for 1 min at 4 °C. After gently removing the aqueous layer, protein pellets were washed with 200 μL cold methanol, spinning down at 18000 g for 1 min at 4 °C, and liquid was gently decanted. After washing twice, pellets were air-dried before boiling with 1X Laemmli buffer.

5. Samples were boiled with 1X Laemmli buffer at 95 °C for 5 min before being loaded onto a 4–20% Tris-HCl gel for SDS-PAGE.

6. In-gel fluorescence scanning was performed using a Typhoon 9400 imager.

7. Gel was then stained by Coomassie blue staining.

3. Label-Free quantitative proteomics analysis of SCFA-reporter Salmonella total cell lysates

3.1. Equipment

37 °C incubator with shaker

Refrigerated centrifuge

Sonic Dismembrator Model 500 (Fisher Scientific)

Water bath sonication

Vortex mixer

Nutating mixture

Vacuum concentrator

Dionex 3000 nano-HPLC coupled to an Orbitrap XL mass spectrometer (Thermo Fisher)

home-made C18 reverse-phase column (75 μm diameter, 15 cm length)

MaxQuant software (https://www.maxquant.org/)

Perseus software (http://www.perseusframework.org/)

−20 °C freezer

3.2. Materials

Salmonella Typhimurium strain 14028S

Miller LB Broth

Miller LB Broth with 300 mM NaCl (SPI-1 inducing LB)

Pentynoate (Alk-3)

PBS buffer

Nonidet P-40

EDTA-free protease inhibitor cocktail (Roche)

Lysozyme

Benzonase (Millipore)

BCA Protein Assay Kit (Thermo)

az-Biotin (Azide-PEG3-biotin conjugate, Sigma)

tris(2-carboxyethyl)phosphine hydrochloride (TCEP)

tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)

t-butanol

DMSO

CuSO₄

Methanol

4% SDS PBS

PBS-T (0.1% tween 20 in PBS)

High Capacity NeutrAvidin agarose (Pierce)

1% SDS PBS

1M Urea PBS

DTT (Sigma)

iodoacetamide (Sigma)

NH₄HCO₃

Trypsin/Lys-C mix (Promega)

custom-made stage-tip containing Empore SPE Extraction Disk (3M)

acetonitrile

formic acid

buffer A (HPLC grade water with 0.1% formic acid)

buffer B (HPLC grade acetonitrile with 0.1% formic acid)

3.3. Protocol

3.3.1. Preparation of Salmonella total cell lysates for proteomics

1. 1:50 dilutions of overnight Miller LB cultures of Salmonella Typhimurium strain 14028S WT were grown in 20 mL SPI-1 inducing LB for 4 h at 37 °C with 220 rpm shaking, with or without alk-3 (4 replicates for each condition).

2. The culture medium was centrifuged at 5000 g for 10 min. Bacteria pellets was re-suspended in 3 mL cold PBS and centrifuged 5000 g 4 °C for 5 min. After re-suspension in 1 mL lysis buffer, bacteria were sonicated for 15 sec with Sonic Dismembrator Model 500 (Fisher Scientific) with 5 sec on and 10 sec off per cycle.

3. Cell lysates were centrifuged at 15000 g for 1 min to remove cell debris and supernatants were collected.

3.3.2. Enrichment of alk-3-labeled proteins

1. Each total cell lysates (~2 mg, tested with BSA assay) was added with 100 μL of click chemistry reagents as a 10X master mix (az-Biotin: 0.1 mM, 10 mM stock solution in DMSO; tris(2-carboxyethyl)phosphine hydrochloride (TCEP): 1 mM, 50 mM freshly prepared stock solution in dH₂O; tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA): (0.1 mM, 2 mM stock in 4:1 t-butanol: DMSO); CuSO₄ (1 mM, 50 mM freshly prepared stock in dH₂O). Samples were mixed well and incubated at room temperature for 1 h.

2. After incubation, samples were mixed with 4 mL cold methanol and incubated at −20°C overnight.

3. Protein pellets were centrifuged at 5000 g for 30 min at 4 °C, pellets were transferred to 2.0 mL centrifuge tube and were washed with 1 mL cold methanol 3 times.

4. After last wash, pellets were let air dried before being re-solubilized in 250 uL 4% SDS PBS with bath sonication. Solutions were diluted with 750 uL PBS, and incubated with 60 uL PBS-T-washed High Capacity NeutrAvidin agarose (Pierce) (500 uL PBS-T-washed twice, 2500 g for 60 s) at room temperature for 1.5 h with end-to-end rotation.

3.3.3. Sample preparation for proteomics

1. Agarose were washed with 500 uL 1% SDS PBS 3 times, 500 uL 1M Urea PBS 3 times, and 500 uL PBS 3 times. Agarose were then reduced with 500 uL 10 mM DTT (Sigma) (50 uL 100 mM to 450 uL PBS) in PBS for 30 min at 37 °C, and alkylated with 500 uL 50 mM iodoacetamide (Sigma) (50 uL 500 mM to 450 uL PBS) in PBS for 30 min in dark.

2. 50 uL NH₄HCO₃ (10 mM) was added to the tube. On-bead proteins were digested with 400 ng Trypsin/Lys-C mix (Promega) at 37 °C overnight with shaking (1000 rpm).

3. Digested peptides were collected (2500 g for 60 s) and lyophilized before being desalted with custom-made stage-tip containing Empore SPE Extraction Disk (3M). Peptides were eluted with 2% acetonitrile, 2% formic acid in dH₂O.

3.3.4. Proteomics analysis of alk-3-enriched proteins

1. Peptide LC-MS analysis was performed with a Dionex 3000 nano-HPLC coupled to an Orbitrap XL mass spectrometer (Thermo Fisher). Peptide samples were pressure-loaded onto a home-made C18 reverse-phase column (75 μm diameter, 15 cm length). A 180-minute gradient increasing from 95% buffer A (HPLC grade water with 0.1% formic acid) and 5% buffer B (HPLC grade acetonitrile with 0.1% formic acid) to 75% buffer B in 133 minutes was used at 200 nL/min.

2. The Orbitrap XL was operated in top-8-CID-mode with MS spectra measured at a resolution of 60,000@m/z 400. One full MS scan (300–2000 MW) was followed by three data-dependent scans of the most intense ions with dynamic exclusion enabled. Peptides fulfilling a Percolator calculated 1% false discovery rate (FDR) threshold were reported.

3. Label-free quantification of alk-3 labeled proteins was then performed with the label-free MaxLFQ algorithm in MaxQuant software as described (Cox et al., 2014).

4. The search results from MaxQuant were analyzed by Perseus. Briefly, the alk-3 labeled sample replicates and control replicates were grouped correspondingly. The results were cleaned to filter off reverse hits and contaminants. Only proteins that were identified in 3 out 4 alk-3 labeled sample replicates and with more than two unique peptides were subjected to subsequent statistical analysis. LFQ intensities were used for measuring protein abundance and logarithmized. Signals that were originally zero were imputed with random numbers from a normal distribution, whose mean and standard deviation were chosen to best simulate low abundance values below the noise level (Replace missing values by normal distribution – Width = 0.3; Shift = 1.8).

5. Significant proteins that were more enriched in alk-3 labeled sample group versus control group were determined by a threshold strategy, which combined t-test p-values with ratio information.

3.3.5. Proteomics analysis of alk-aspirin-enriched proteins

We found that treatment of Salmonella with alk-aspirin (1 mM) at 37 °C for 4 h can significantly affect Salmonella gene transcription and inhibits Salmonella growth. To explore alk-aspirin modified proteins by proteomics, we modified the bacteria and small molecules incubation conditions as following: 1:50 dilutions of overnight Miller LB cultures of Salmonella Typhimurium strain 14028S WT were grown in 20 mL SPI-1 inducing LB for 3 h at 37 °C with 220 rpm shaking. Cultures were incubated with 0.5 mM of alk-aspirin in DMSO or DMSO as control for another 1 h at 37 °C with 220rpm shaking. Other protocol for in-gel profiling and label-Free quantitative proteomics analysis of alk-aspirin modified Salmonella total cell lysates are same as described above for Alk-3 reporter.

4. CRISPR-Cas9 editing and epitope-tagging of proteins in Salmonella

4.1. Equipment

37 °C incubator with shaker

Refrigerated centrifuge

Gene Pulser II (Bio-Rad)

PCR Thermal Cyclers

−80 °C freezer

−20 °C freezer

4.2. Materials

Salmonella Typhimurium strain 14028S

Miller LB Broth

glycerol

pKD46 (Datsenko and Wanner, 2000)

2 mm Gap Sterile Electroporation Cuvette

carbenicillin (100 ug/mL) LB agar plates

arabinose

pCas9 (Jiang et al., 2013)

Single-stranded DNA (ssDNA) editing template

SOC medium (Sigma)

chloramphenicol (25 ug/mL) LB agar plates

plain LB agar plates (without antibiotic)

4.3. Protocol

1. Overnight culture of S. Typhimurium strains were diluted 1:50 to 20 mL fresh LB medium, and were grown at 37 °C in a shaking incubator at 220 rpm until the OD600 reached 0.5–0.7. Cells were pelleted at 5,000 g for 10 min at 4 °C, and washed twice with 10 mL ice-cold 10% glycerol. Cell pellets were resuspended in 100 μL 10% glycerol, and aliquoted as 50 μL per tube, then stored in −80 °C. Electrocompetent parent S. Typhimurium strains were transformed with pKD46 via electroporation with Gene Pulser II (Bio-Rad) at 2.5 kV and 25 μF in a 2-mm cuvette followed by addition of warm SOC medium (950 μL). Bacteria was recovered at 30 °C for 1 h with 220 rpm shaking and selected on carbenicillin agar plates at 30 °C overnight.

2. The resulting S. Typhimurium pKD46 strains were made into electrocompetent cells after being grown at 30 °C with 0.2% arabinose and carbenicillin until the OD600 reached 0.5–0.7. S. Typhimurium pKD46 electrocompetent cells were transformed with 2 μL pCas9 (~100ng) and 2 μg ssDNA editing template followed by addition of warm SOC medium (950 μL). Bacteria was recovered at 37 °C for 2 h with 220 rpm shaking.

3. Then resulting bacteria were concentrated to 200 μL by centrifugation (14000 g x 1 min) and selected on chloramphenicol agar plates at 37 °C overnight. Colonies on the plate were streaked onto new chloramphenicol agar plates, and colonies from the new plates were randomly picked for colony PCR to confirm editing (50%~100% efficiency). Successfully edited colonies were further confirmed by Sanger sequence.

4. The pCas9 was cured by growing bacteria in plain LB agar.

5. Validation of SCFA-reporter protein modification in Salmonella

5.1. Equipment

37 °C incubator with shaker

Refrigerated centrifuge

Water bath sonicator

Microplate readers (BioTek)

Vortex mixer

−80 °C freezer

−20 °C freezer

Nutating shaker

Block heater

Criterion vertical electrophoresis cell (Bio-Rad)

Typhoon 9400 imager (Amersham Biosciences)

ChemiDoc Imaging System (Bio-Rad)

Trans-Blot Turbo Transfer System (Bio-Rad)

Orbital shaker

5.2. Materials

Salmonella Typhimurium strain 14028S

Miller LB Broth

Miller LB Broth with 300 mM NaCl (SPI-1 inducing LB)

Pentynoate (Alk-3)

PBS buffer

Nonidet P-40

EDTA-free protease inhibitor cocktail (Roche)

Lysozyme

Benzonase (Millipore)

BCA Protein Assay Kit (Thermo)

Pierce™ Anti-HA Magnetic Beads (Thermo)

az-Rho (Rangan et al., 2010)

az-Biotin (Azide-PEG3-biotin conjugate, Sigma)

tris(2-carboxyethyl)phosphine hydrochloride (TCEP)

tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)

High Capacity NeutrAvidin agarose (Pierce)

t-butanol

DMSO

CuSO₄

0.45 μm nitrocellulose membrane (Bio-Rad)

non-fat milk

anti-HA rabbit antibody H6908 (Sigma)

goat polyclonal anti-rabbit HRP ab97051 (Abcam)

Clarity Western ECL substrate (Bio-Rad)

1X Laemmli buffer

4–20% Tris-HCl gel (Bio-Rad)

Coomassie Stains (Bio-Rad)

5.3. Protocol

5.3.1. In-gel fluorescence analysis of alk-3 protein modification in Salmonella

1. Salmonella Typhimurium strain 14028S with HA tag was inoculated in 4 mL Miller LB. Bacteria was cultured overnight at 37 °C with 220 rpm shaking.

2. 1:50 dilutions of overnight culture of Salmonella Typhimurium strain 14028S with tag were grown in 4 mL SPI-1 inducing LB (with or without Alk-3) for 4 h at 37 °C with 220 rpm shaking.

3. S. Typhimurium cells were pelleted at 15000 g for 1 min, and pellets washed with cold PBS once.

4. The pellet was lysed with 200 μL lysis buffer (phosphate-buffered saline (PBS) containing 0.5% Nonidet P-40, 1X EDTA-free protease inhibitor cocktail (Roche), 0.5 mg/mL lysozyme (in dH₂O) (Sigma), and 1:1,000 dilution of Benzonase (Millipore)). After re-suspension, pellets were sonicated for 10 sec for 3 times, then were incubated on ice for 30 min. Cell lysates were centrifuged at 8200 g for 5 min to remove cell debris and supernatants were collected.

5. Protein concentration was estimated by BCA assay with BCA Protein Assay Kit (Thermo).

6. 250 μg of each total cell lysates were immunoprecipitated with 20 μL PBS-T-washed Pierce™ Anti-HA Magnetic Beads (Thermo Scientific). Samples were mixed well and incubated at 4 °C for 2 h with end-to-end rotation.

7. After samples were washed with 500 μL PBS-T 3 times, 44 μL of PBS-T (PBS with 0.1% Tween-20) was added to each sample. 5 μL of click chemistry reagents as a 10X master mix mentioned above were added to each sample. After incubation at room temperature for 1 h, samples were washed with 500 μL PBS-T 3 times.

8. Samples were boiled with 2X Laemmli buffer at 95 °C for 5 min before being loaded onto a 4–20% Tris-HCl gel (Bio-Rad) for SDS-PAGE. In-gel fluorescence scanning was performed using a Typhoon 9400 imager (Amersham Biosciences) or ChemiDoc Imaging Systems.

9. Proteins were then transferred onto 0.45 μm nitrocellulose membrane (Bio-Rad) with Trans-Blot Turbo Transfer System (Bio-Rad) at 25 V for 30 min.

10. The membrane was blocked with 5% non-fat milk in PBS-T for 60 min, and 1:5,000 anti-HA rabbit antibody H6908 (Sigma) was added to solution before incubating membrane at 4 °C overnight. The membrane was washed with PBS-T 3 times, and incubated with 1:10,000 goat polyclonal anti-rabbit HRP ab97051 (Abcam) in PBS-T with 5% non-fat milk at room temperature for 1 h.

11. The membrane was washed with PBS-T 3 times, and imaged with Clarity Western ECL substrate (Bio-Rad) and ChemiDoc XRS+ System (Bio-Rad).

5.3.2. Validation of alk-aspirin protein labeling by immunoblotting

Alk-butyrylated aspirin chemical reporter (alk-aspirin) was applied as a chemical reporter to explore butyryl-salicylic acid labeling proteins in Salmonella. Here, we present the validation of alk-aspirin protein labeling by immunoblotting.

1. 1. From the alk-aspirin-treated or control total cell lysates prepared as described above, 90 μL of each total cell lysates (~100 μg) was added with 10 μL of click chemistry reagents as a 10X master mix (Az-biotin: 0.1 mM, 10 mM stock solution in DMSO; tris(2-carboxyethyl)phosphine hydrochloride (TCEP): 1 mM, 50 mM freshly prepared stock solution in dH₂O; tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA): (0.1 mM, 2 mM stock in 4:1 t-butanol: DMSO); CuSO₄ (1 mM, 50 mM freshly prepared stock in dH₂O). Samples were mixed well and incubated at room temperature for 1 h.

2. 2. After incubation, samples were mixed with 200 μL cold methanol and incubate at −20 °C overnight.

3. 3. Sample proteins were precipitated at 18000 g for 15 min at 4 °C. After gently removing the aqueous layer, protein pellets were washed with 200 μL cold methanol, spinning down at 18000 g for 15 min at 4 °C, and liquid was gently decanted. After last wash, pellets were let air dried (37 °C for 1 h) before being re-solubilized in 100 uL 4% SDS PBS with bath sonication (5 uL solution was kept for “input” before enrichment).

4. 4. Solutions were then diluted with 300 uL PBS, and incubated with 20 uL PBS-T-washed High Capacity NeutrAvidin agarose (Pierce) (500 uL PBS-T-washed twice, 2500 g x 60 s) at room temperature for 1 h with end-to-end rotation. The agarose was washed with 500 uL 1% SDS PBS 3 times, 500 uL 1M Urea PBS 3 times, and 500 uL PBS 3 times. Samples were boiled with 2X Laemmli buffer 95 °C for 5 min before being loaded onto a 4–20% Tris-HCl gel (Bio-Rad) for SDS-PAGE. Equal loading is validated by immunoblotting of input samples before enrichment (“Input”).

6. Summary and outlook

Metabolites from gut microbiota can directly regulate pathogen virulence via diverse mechanisms. To investigate the molecular mechanism, here we describe targeted and proteomic protocols to characterize the metabolite-modified proteins in Salmonella. Metabolite reporters such as alk-3 as well as its derivatives bearing an alkyne chemical handle facilitated the analysis of covalently-modified proteins. To investigate non-covalent metabolite-protein interactions in bacteria, photoreactive functional groups such as diazirines or benzophenones can be employed. The application of metabolic and photoaffinity chemical reporters should enable additional mechanistic studies of metabolites in bacteria and mammalian cells in the future.