Genevieve C. Van de Bittner1, Alex Apffel1,Kristin B. Bernick1, Manuel Gomez2, Dustin
Chang1, Thu T.A. Nguyen1, Brian P. Smart1,Reid Brennen1, Christine Miller3, StevenFischer3, Laurakay Bruhn1

A Room Temperature Metabolism Quenching Method
for Automated Metabolomics Sample Preparation

Metabolomics
2019
Poster #: 293

1Agilent Research Labs, 2LSAG Mass Spec

Division, 3ACG LSSM - Marketing and Digital,
Agilent Technologies, Santa Clara, CA

Introduction

K562 Leukemia Cell Line

13C -Glutamate from 13C -Glutamine in Cell Extracts
55

Peak Area (Counts)

5000000

RT Solution

PBS

HTB123 Breast Cancer Cell Line0RT Solution

PBS

A single solution (RT solution) was used
for room temperature cell lysis andmetabolism quenching. Trypan bluestaining indicates the RT solution lyses
mammalian suspension cells. Brightfield
images show cells not lysed (not stained)
or lysed (stained).

5 13C carbons

5 13C carbons

ATP is Stable in Cell Lysates Generated with RT Solution
Initial Samples

ATP (μM)

804060304020

Diluted Samples
6010X
Dilution

201000Unquenched

RT Solution

5 min

Equivalent amounts of ATP were extracted from K562 cells by the RT solution
at room temperature and a traditional extraction solution, 2:2:1 MeOH:ACN:
H2O, 0.1% FA, at -20°C.1 Lysed, unquenched cells exhibit lower ATP levels.

50403020100PBS

Traditional -20°CSolution

60 min

RT Solution
120 minPBS

240 min5 min

60 min

RT Solution
120 min

240 minATP extracted with the RT solution is stable at room temperature for up to four hours, while ATP extracted intoPBS via probe sonication is not stable. ATP extracted with the RT solution remains stable at room temperature
after a 10-fold dilution into PBS. For diluted samples, the measured ATP concentration was multiplied 10-fold.

6. Sample Cleanup to Remove Lipids
Captiva EMR-Lipid for Lipid Removal

Optimized Conditions for Metabolite Recovery from Captiva EMR-Lipid Plate
Fraction Recovered

1.4

Sorbent

Small Molecule

2. Wash Cells (optional)

1.2
1.0

80%
Recovery

0.8
0.6
0.4
0.2
0.0

Lipid

Optimized

EtOH_ACN_50%_water

Lipid removal improves LC/MS performance by reducing
matrix effects caused by phospholipids2. The Captiva EMRlipid (Enhanced Matrix Removal) sorbent removes lipidsbased on size exclusion and hydrophobic interactions.

Starting Conditions

EtOH_ACN_18%_water

Optimized sample conditions were created for maximizing metabolite recovery from the Captiva EMR-lipid plate. The
optimized conditions have an average 80% recovery of central carbon and other metabolites, as measured by LC/MS.3 Similar
metabolite recoveries are obtained with cell lysates made using the RT solution.

Captiva EMR-Lipid Plate Removes Lipids under Optimized Metabolite Recovery Conditions
FA 2PIPGSM

EtherPCPE

EMR-treated

FA 1

non-EMR-treated

5. Adjust Solvent CompositionCompatible with Bravo Platform

96-Well Plate Format

6. Sample Cleanup to Remove Lipids

Metabolism quenching was monitored in K562 cell lysate using isotope
tracing with 13C5-glutamine. Left: schematic of the TCA cycle, indicating
the flow of carbon-13 atoms as they enter the cycle, downstream of 13C5glutamine. indicates compounds monitored. Above: 13C5-glutamate
detected in cell lysate from cells lysed and quenched with solutions
containing 13C5-glutamine. The RT solution performs better than all otherlysis and quenching solutions, including room temperature 2:2:1
MeOH:ACN:H2O, 0.1M FA (221).1 RT = room temperature.

4 13C carbons

Room Temperature Solution Extracts ATPEMR

4. Precipitate Proteins

500000Background 13C5-Glutamate

Workflow

3. Lyse Cells and Quench Metabolism
at Room Temperature (RT Solution)

4000000
1000000

4 13C carbons• Room temperature lysis and quenching are
more easily automated than traditional
workflows that utilize hot or cold quenchingsolutions

1. Count and Harvest Cells

4500000

4 13C carbons• Room temperature workflow is simpler andsafer than traditional workflows• Includes the integration of the Agilent Captiva
EMR-Lipid plate which captures lipids for
improved LC/MS performance

Isotope Tracing Verifies Metabolism Quenching at Room Temperature

ATP (μM)

Advantages of Room Temperature Lysis andQuenching:Room Temperature Cell Lysis

ATP (μM)

Many existing methods for cell-based metabolomic
sample preparation require low-temperature
metabolism quenching procedures, which prevent
interconversion of metabolites in a rapid, controlledmanner. The low temperatures required to quench
metabolism often utilize cold liquids, such as liquid
nitrogen, which are difficult to handle and store.
Additionally,lowtemperaturescomplicate
automation of metabolism quenching steps, since
they promote collection of condensation on roboticcomponents. This condensation can hinder robotic
movement and cause rusting. We developed amass-spectrometry-compatible method for room
temperature
metabolism
quenchingandsimultaneous cell lysis to avoid the issues
associated with low temperature quenching.

3. Lyse Cells and Quench Metabolism at Room Temperature (RT Solution)

Captiva EMR-lipid 96-well plates have been utilized in theBravo Metabolomics Sample Prep Platform, which
automates metabolomics sample preparation for plasmasamples.

7. Dry Samples

Captiva EMR-lipid plate removes lipids from cell lysate samples prepared using the RT solution and conditions optimized formetabolite recovery from the Captiva EMR-lipid plate. Fatty acids are incompletely removed while PI, PG, SM, PC, and PE lipids
are completely removed from the cell lysate samples.

9. Analyze Samples by LC/MS
Preliminary Untargeted LC/MS Comparison of the Room Temperature Workflow to a Traditional Workflow
938 Unique Peaks

8. Reconstitute Samples9. Analyze Samples by LC/MS

Log2 Scaled
Peak Area
21.5

Traditional Method

17.1

Room Temp Method
ManualConclusions• We developed a room temperature method
for mammalian cell lysis and metabolism
quenching that is more amenable toautomation than conventional methods.
• We optimized conditions for clean-up of celllysates using the Captiva EMR-lipid sorbent.
• Initial comparison shows similar numbers ofpeaks are detected in samples prepared with
our room temperature method compared toa traditional method.
• Work is ongoing to automate the room
temperature method on the Bravo liquidhandling platform and quantify performancecompared to traditional methods.

12.7
8.2

Room Temp Methodwith Prototype Automation Plates

3.8Samples were prepared from K562 cells using either the developed room temperature workflow (manual or with plates from a prototype automated workflow) or a traditional workflow utilizing-20°C temperatures. Cell samples were analyzed by LC/MS.3 Data analysis was completed using Agilent Profinder and Mass Profiler Professional software. All peaks present in significantlevels in blank injection samples were removed from the data. A similar number of peaks are found using all methods. Differences between the methods are under investigation.

Planned Room Temperature Automated Workflow
Mammalian Cells

Cell
Transfer

Bravo

Nitrogen
Evaporator

References
1)

Lu, W., Su, X., Klein, M.S., Lewis, I.A., Fiehn, O., and Rabinowitz, J.D.
Metabolite Measurement: Pitfalls to Avoid and Practices to Follow. Annu.
Rev. Biochem. 2017, 86, 277-304.

2)

Zhao, L. and Lucas, D. Efficiency of Biological Fluid Matrix Removal
Using Agilent Captiva EMR-Lipid Cleanup. Agilent Technologies
Application Note.

3)

Hartman, T. E., Rhee, K. Y., Dai, Y. Metabolomics Analysis ofTuberculosis Drug Activity Using an Agilent 6545 Q-TOF LC/MS. Agilent
Technologies Application Note.

LC/MS

SampleTransferor

Acknowledgements: We would like to thank Dan Cuthbertson at Agilent
Technologies for assistance with the MPP analysis.
Analyze Samples

For Research Use Only.
Not for use in diagnostic procedures.
RA44448.4315277778