Investigating Antibiotic Mode ofAction Using Targeted Metabolomics
with Ion-pairing Reversed-phase
LC/Q-TOF
Application NoteMetabolomics

Authors

Abstract

Amy A. Caudy1,2, Soumaya Zlitni1, andMetabolomics is a growing analytical field characterizing the biochemical contents

Adam P. Rosebrock1,2

and reactions of the cell. Determining metabolic state requires separation and mea-

1 Donnelly Centre for Cellular andBiomolecular Researchsurement of a diverse set of small molecules. We have developed an ion-paired
reversed-phase chromatographic method that enables the robust separation ofanionic and hydrophobic metabolites. This method enables simultaneous analysis of

2 Department of Molecular Genetics

University of Toronto

hundreds of compounds including amino acids, citric acid cycle intermediates, andother carboxylic acids, nucleobases, nucleosides, phosphosugars, and fatty acids.
When used in conjunction with accurate mass TOF/Q-TOF or triple-quadrupole
mass spectrometers, this separation enables rapid, robust, and reproducible quantitation of a large array of compounds in central metabolism across a wide range ofconcentrations. Using this method, we measured metabolite levels following treatment of the gram negative bacteria E. coli with the antibiotic glyphosate. Analyzingchanges in metabolite levels revealed the mechanism of action of glyphosate,demonstrating the power of metabolomics for direct biochemical phenotyping, andillustrating its potential use in uncovering novel mechanisms of action.

Introduction
H

A major goal of metabolomic analysis is the simultaneousquantitation of molecules possessing a variety of functionalgroups and ranging widely in mass and hydrophobicity. Many
chromatographic modalities including normal phase, HILIC,
and reversed-phase have been applied to metabolomic analysis, but are generally suboptimal due to poor selectivity, poor
run-to-run reproducibility, or nonretention of polar metabolites. Ion-paired reversed-phase analysis has the advantage ofproviding retention of polar compounds on a conventional
reversed-phase column by functionalization of the stationary
phase with a bifunctional ion-pairing agent. These agents
possess an ionizable moiety, and one or more hydrophobicelements. The ionizable group of the ion-pairing agent interacts with polar compounds, while the hydrophobic groupinteracts with a reversed-phase column stationary phase suchas C18 (Figure 1).

Agilent Extend C18HOSiO SiOHOPOH

Figure 1.

The tertiary amine tributylamine provides a cationicfunctionalization of the reversed-phase packing with which
many acidic metabolites can interact. Selectivity of conventional reversed-phase analytes, including hydrophobic
metabolites, is maintained in the presence of tributylamine.

CountsOOOOH OH

11.354
2.556

0234

567Acquisition time (min)89101112

11.391Counts432101

Figure 2.

234567Acquisition time (min)

POH

NOH
Tributylamine

Ion pairing with tributylamine functionalizes C18 side chains,
permitting retention of polar compounds like phosphosugars inaddition to nonpolar compounds that are typically retained inreversed phase analysis.

×105
1 A
1OOH OH

Previous ion-paired reversed-phase methods have been limited in their application to large or extended sample sets byprogressive chromatographic drift over multiple sample injections. Our approach uses a physically robust Agilent Extend
C18 column packing in a UPLC configuration that permitsbackflushing during column regeneration. We find that thisoff-line backflushing and regeneration provides superior
system performance and excellent run-to-run reproducibility(Figure 2). While this regenerative backflushing is possible ona single pump system, we present a method that makes efficient use of the connected mass spectrometer by using a pairof identical columns and alternating column regeneration.

This application note describes an ion-paired reversed-phase
LC/MS metabolomics analytical method specificallydeveloped for long-term reliability and reproducibility. Thisapproach can be used to perform relative quantitation ofendogenous cellular metabolites, and extended to semiquantitative or quantitative analysis through the use of spike-in andisotopically labeled internal controls.

×1055 B

HHO

89101112

Backflushing during column regeneration increases retention time stability. Repeat injections of thesame sample without backflushing (A) and with backflushing (B).

2The LC system includes two binary pumps connected to twocolumns through a two-position, 10-port valve to allow one
column to be run on an analytical gradient to the mass
spectrometer, while the other column is backflushed andregenerated off-line (Figure 3). The backflush provides superior long-term stability of retention time compared toconventional forward-flow column regeneration (Figure 4).
The method used the Agilent RRHD Extend-C18 column,
which features high pH stability, potentially improving column
life in the presence of the strongly basic ion-pairing agent.

Retention time for 62 individual sample injections

20Agilent 1290 Infinity Binary
from injector

21105093Column 28Column 1
4756Agilent 1260 Infinity Binary
Aux pump

To mass spectrometer

Figure 3.

15010Retention time after >4,000 injections (seven months of use)

Waste

A
62 Different samples, 3 days

Alternate column regeneration permits higher throughput
analysis. Using a 2-position, 10-port valve, one column isconnected to the sample injector while the second is connected to a binary pump for column regeneration. In thesecond position, the first column is regenerated, while thesecond is used for sample analysis. The flow from theregeneration pump is backflushed through the column toprovide superior column cleaning.

We demonstrate this method in the metabolic profiling of thegram negative bacterium E. coli exposed to glyphosate, anaminophosphonate analogue of glycine. Better known as thewidely used herbicide Round-Up, glyphosate inhibits
5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, acritical step in the synthesis of chorismate and de novo
biosynthesis of all aromatic amino acids1. Many higher animals require dietary intake of aromatic amino acids due to theabsence of the de novo biosynthetic pathways. Glyphosate
has low toxicity in mammals, and has been considered as acandidate antibiotic. We examined themetabolic consequences of glyphosate treatment of E. coli todemonstrate the potential of LC/MS metabolomics to measure metabolic changes induced by small molecule treatment,
identify mode of action, and serve as a platform to assist in
drug discovery and preclinical studies.

2020

B
Aliquoted sample,
seven months of active column use(> 4,000 injections)

15

10500

Figure 4.

351015
Average retention time (minutes) across 62 samples

51015
Retention time (minutes) for fresh column

20Chromatographic retention time is stable over many injections.
Shown are chromatographic retention times for a set of known
compounds. A) Retention time deviation versus retention time for
62 different biological samples (extracts of yeast cells) analyzed
over the course of three days. B) Retention times for analytes inthe same sample, analyzed before and after an intervening
4,000 injections of biological samples.

Experimental

Instrumentation

Sample preparation

An Agilent 6550 Quadrupole Time-of-Flight LC/MS
consisting of:

For preparation of metabolite extracts, E. coli MG1655 cultures were grown at room temperature in 24-well blocks containing 5.5 mL M5 minimal medium per well. At the mid-log
phase, glyphosate or an equal volume of vehicle was added tothe culture. A 5.0 mL volume of culture was transferred to a
25 mm diameter Pall Nylaflo 0.2-µm nylon membrane filter ona Hoefer FHV-225 manifold and cells were collected byvacuum filtration. Filters containing cells were immediatelytransferred to a 15-mL Falcon tube containing 1.2 mL ofextraction solvent prechilled to –20 °C. The tube was vortexed
to resuspend the cells in solvent, and immediately returned to–20 °C. Preparation of metabolite extracts from filtration tostorage took ~12 seconds per sample. The extraction solvent
consisted of a 40:40:20 ratio of acetonitrile, methanol, andwater cooled to –20 °C. Optical density was measured from asmall sample of culture at the time of metabolite extraction asa proxy of culture concentration. Cells in the extraction
solvent were lysed by three freeze-thaw cycles alternating
between –80 °C and –20 °C. Following the final thaw at–20 °C, filters were removed from the tube and insoluble
material was collected by centrifugation at 4,000xg for
15 minutes at 4 °C. Supernatant containing extracted metabolites was transferred to a fresh polypropylene tube, and driedin a TurboVap LV evaporator under dry N2. Dried metabolite
extracts were resuspended by vortexing in a volume ofHPLC-grade H2O adjusted to the cell density determined byoptical density at the time of cell harvest. Following resuspension, samples were briefly centrifuged to collect material,
transferred to a Pall Acroprep 0.2-µm multiwell hydrophilic
polypropylene filter plate, and vacuum-filtered into a
polypropylene receiver plate. Filtered extracts were loaded
into conical polypropylene HPLC vials, and placed in a 4 °C
autosampler for LC/MS analysis.

• Agilent 1290 Infinity Binary UHPLC pump
• Agilent 1260 Infinity Binary pump
• Agilent 1290 Infinity Thermostatted Column Compartment• Agilent 1290 Infinity HTS Autosampler.
An Agilent 6550 accurate-mass Q-TOF mass spectrometerwith an Agilent Jet Stream dual ionization source were usedto acquire data in high resolution mode at 4 GHz at two
spectra per second.
LC Conditions
ColumnAgilent RRHD Extend-C18, 2.1 × 100 mm, 1.8 µm

Mobile phaseA) Water:methanol 97:3 with 10 mM tributylamineand 15 mM acetic acidB) Methanol for analytical separation,acetonitrile for regeneration

Gradient

Analytical phase: Methanol in channel B,
flow rate 0.25 mL/min
1% B at 0 minutes
1% B at 2.5 minutes
20% B at 7.5 minutes
55% B at 13 minutes
99% B at 20 minutes
99% B at 21 minutes
1% B at 22.9 minutes
1% B at 23 minutes
Regeneration phase: Switch 2-position, 10-portvalve to second pump with acetonitrile inchannel B. This program runs while an analyticalgradient is running on the other column.
1% B at 23 minutes, 0.2 mL/min
99% B at 26 minutes, 0.2 mL/min
99% B at 27 minutes, 0.4 mL/min
99% B at 33.9 minutes, 0.4 mL/min
99% B at 34 minutes, 0.25 mL/min
1% B at 36 minutes, 0.25 mL/min
1% B at 46 minutes, 0.25 mL/min

Column temperature

37 °C

Autosampler temperature 4 °C
Injection volume42 µL

MS Conditions

Glyphosate treatedIonization mode

Jet Stream

Ionization polarity

Negative

Gas temperature

150 °C

Drying gas

14 L/min

Nebulizer pressure

45 psiControl

Sheath gas temperature 325 °C
Sheath gas flow

12 L/min

ESI Capillary voltage

2,000 VNozzle voltage

300 V

Fragmentor voltage

340 V

Octopole 1 RF voltage

600 V

Scan range

m/z 25–1,100

Reference masses

m/z 112.9855 (trifluoroacetic acid),
1033.9981 (HP-0921)

Data analysis
The chromatographic retention times of known metabolites
were determined by LC/MS analysis of neat standards togenerate an accurate mass/retention time database. We
used Agilent MassHunter Profinder in Batch Molecular
Feature Extraction mode to extract features in this databasefrom biological sample d files. .CEF files from Profinder wereimported into Agilent MassHunter Mass Profiler Professional
for preprocessing, hierarchical clustering, and statisticalanalysis. Agilent MassHunter PathwayArchitect was used todisplay changes in metabolite levels on BioCyc pathways.

Results and Discussion
E. coli cells were grown to log phase, treated with glyphosate,and harvested for metabolite extraction. The quantity of eachmetabolite was determined by integrating an extracted ion
chromatogram (EIC) centered at the known retention time ofa metabolite standard, and the area under the curve was calculated and the blank subtracted. These values of abundancefor each metabolite were log2 transformed and centered to a
median change of zero to highlight differences among thesamples for each metabolite. The metabolite abundanceswere clustered, and the log2 changes in metabolite abundances were displayed as a heatmap, in which red represents
an increase in metabolite compared to the median value ofthat metabolite for all samples, and blue represents adecrease in the value of that metabolite for the samples.

-6Figure 5.

50log2 fold-change+6Levels of known metabolites following treatment with glyphosate
or vehicle control. Data were extracted using Agilent MassHunter
Profinder in Batch Molecular Feature Extraction mode and analyzed using Agilent MassHunter Mass Profiler Professional.
Levels for each metabolite are presented as log2 transformed,
mean-subtracted values to identify changes between conditions.

Although EPSP levels are not directly measured and two enzymatic steps exist between 3-phosphoshikimate and chorismate, the resulting changes in up- and down-stream metabolite pools point to disruption in the pathway known to be thetarget of glyphosate, centered around the established targetof action, EPSP-synthase. Even without targeted methods foranalysis for each compound in the pathway, we were able toidentify the correct target of glyphosate treatment.

Targeted metabolomic analysis reveals a number ofsignificantly changing metabolites between treated andcontrol cells (Figure 5).
Large increases in shikimate and 3-phosphoshikimate wereobserved following glyphospate treatment, while chorismate
and all three aromatic amino acids were significantly
decreased in concentration following treatment (Figure 6).

EPSP-Synthase:target of Glyphosate

Accumulating metabolites
DepletedD
pl d mmet
metabolites
et b li-60+6log2 fold-change

Glyphosate
treated

Untreated
control

replicate 1
replicate 2
replicate 3
replicate 4
replicate 5
replicate 6

Figure 6.

Effects of glyphosate treatment are visible as a concerted change in many intermediate and end-product metabolites. Using Pathway Architect andAgilent MassHunter Mass Profiler Professional, aromatic amino acid biosynthesis was identified as the pathway most significantly affected in thisexperiment. Individual metabolite measurements for glyphosate-treated and control-treated cells are shown. Intermediates upstream ofEPSP-synthase accumulate upon treatment, while downstream intermediates and end products are highly depleted (with several more than
4,096x decreased).

6Conclusions
We have developed a reversed-phase ion-pairing method thatprovides a platform for the robust analysis of a wide range ofendogenous anionic metabolites. Regenerative backflushingand the use of a ternary solvent for regeneration of theAgilent Extend C18 RRHD columns provides superior
long-term performance compared to other ion-paired
reversed-phase analytical methods, including stable retention
times and consistently high quality peak shape, and enables
long column life with minimal sample preparation.
The highly reproducible retention times achieved through thisanalytical method enable targeted metabolomic analysis bynature of minimal chromatographic drift. In addition to use on
an accurate mass instrument, we have deployed this chromatography on the triple quadrupole platform, where retention time consistency enables tight windows for scheduling ofa large number of MRM transitions.
Although targeted analyses are presented here, this method
is readily applicable for full-scan, untargeted analysis andidentification of novel metabolites.

Reference
1. H. C. Steinrücken, N. Amrhein. “The herbicide glyphosate
is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate synthase” Biochem. Biophys. Res. Commun. 94,
1207-1212, (1980).

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