Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Summary

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Abstract

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with ¹³C-aniline, while compounds in the sample are derivatized with ¹²C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Introduction

Cell-free protein synthesis (CFPS) is a promising platform for manufacturing of proteins and chemicals, an application that has traditionally been reserved for living cells. Cell-free systems are derived from crude cell extracts and eliminate the complications associated with cell growth¹. In addition, CFPS allows for direct access to metabolites and the biosynthetic machinery without the interference of a cell wall. However, a fundamental understanding of the performance limits of cell-free processes has been lacking. High-throughput methods for metabolite quantification are valuable for the characterization of metabolism and are critical for the construction of metabolic computational models^2,3,4. Common methods used to determine metabolite concentrations include nuclear magnetic resonance (NMR), Fourier transform-infrared spectroscopy (FT-IR), enzyme-based assays, and mass spectrometry (MS)^5,6,7,8. However, these methods are often limited by their inability to efficiently measure multiple compounds at once and often require a sample size greater than typical cell-free reactions. For example, enzyme-based assays can often only be used to quantify a single compound in a run, and are limited when the sample size is small, such as in cell-free protein synthesis reactions (typically run on a 10-15 μL scale). Meanwhile, NMR requires a high abundance of metabolites for detection and quantification⁵.  Toward these shortcomings, chromatography methods in tandem with mass spectrometry (LC/MS) provide several advantages, including high sensitivity and the capability of measuring multiple species simultaneously⁹; however, the analytical complexity increases considerably with the number and diversity of species being measured. It is important, therefore, to develop methods that fully realize the high-throughput potential of LC/MS systems. Compounds in a sample are separated by liquid chromatography and identified through mass spectrometry. The signal of the compound depends on its concentration and ionization efficiency, where the ionization can vary between compounds and may also depend on the sample matrix.  

Achieving the same ionization efficiency between the sample and standards is a challenge when using LC/MS to quantify analytes. Further, quantification becomes more challenging with metabolite diversity due to signal splitting and heterogeneity in proton affinity and polarity¹⁰. Lastly, the co-eluting matrix of the sample can also affect the ionization efficiencies of the compounds. To address these issues, metabolites can be chemically derivatized, increasing the separation resolution and sensitivity by LC/MS systems, while simultaneously decreasing signal splitting in some cases^10,11. Chemical derivatization works by tagging specific functional groups of metabolites to adjust their physical properties like charge or hydrophobicity to increase ionization efficiency¹¹. Various tagging agents can be used to target different functional groups (e.g., amines, hydroxyls, phosphates, carboxylic acids, etc.). Aniline, one such derivatization agent, targets multiple functional groups at once, and adds a hydrophobic component into hydrophilic molecules, thereby increasing their separation resolution and signal¹². To address the co-eluting matrix ion suppression effect, Yang and coworkers developed a technique based on Group Specific Internal Standard Technology (GSIST) labeling where standards are tagged with ¹³C aniline isotopes and mixed with the sample^12,13. The metabolite and corresponding internal standard have the same ionization efficiency since they co-elute, and their intensity ratio can be used to quantify the concentration in the experimental sample.

In this study, we developed a protocol to detect and quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method is based on the GSIST approach, where we used ¹²C-aniline and ¹³C-aniline to tag, detect, and quantify metabolites using reversed-phase LC/MS. The linear range of all compounds spanned 2-3 orders of magnitude with an average correlation coefficient of 0.988. Thus, the method is a robust and accurate approach to interrogate cell-free metabolism, and possibly whole-cell extracts. 

Protocol

1. Preparation of reagents for aniline tagging

1. Prepare a 6 M aniline solution at pH 4.5. Working in a hood, combine 550 µL of aniline with 337.5 µL of LCMS grade water and 112.5 µL of 12 M hydrochloric acid (HCl) in a centrifuge tube. Vortex well and store at 4 °C.
   NOTE: Aniline can be stored at 4 °C for 2 months.
   CAUTION: Aniline is highly toxic and should be worked with in a fume hood. Hydrochloric acid is highly corrosive
2. Prepare a 6 M ¹³C aniline solution at pH 4.5. Combine 250 mg of ¹³C₆-aniline with 132 µL of water and 44 µL of 12 M HCl. Vortex well and store at 4 °C.
3. Prepare 200 mg/mL N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) solution. Dissolve 2 mg of EDC in 10 µL of water for every sample to be tagged and vortex well.
   NOTE: EDC solution should be prepared the same day as the reaction. EDC acts as a catalyst for the derivatization of compounds with aniline¹².

2. Preparation of standards

1. Make separate stock solutions of all compounds dissolved in LC/MS grade water (Table 1).
2. Preparation of internal standard stock solution
   1. Combine all compounds except for nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), acetyl coenzyme A (ACA), and glycerol 3-phosphate (Gly3P), with the appropriate volumes to create a 2 mM stock solution of all compounds.
3. Combine NAD, NADP, FAD, ACA, and Gly3P with the appropriate volumes to create a 2 mM stock solution.

3. Preparation of sample (Figure 1)

1. Quench and precipitate the proteins in a cell-free protein synthesis reaction by adding an equal volume of ice-cold 100% ethanol to the reaction. Centrifuge the sample at 12,000 x g for 15 min at 4 °C. Transfer the supernatant to a new centrifuge tube.
   NOTE: Samples can be stored at -80 °C at this point and analyzed at a later time

4. Labeling reaction  

1. Labeling sample with ¹²C-aniline solution
   1. Transfer 6 µL of sample into a new centrifuge tube and bring the volume to 50 µL with water.
      NOTE: Volume sample size may depend on the specific CFPS reaction.
   2. Add 5 µL of 200 mg/mL EDC solution.
   3. Add 5 µL of ¹²C-aniline solution.
      NOTE: The aniline solution separates into two phases. Mix well before adding to the reaction.
   4. Vortex the reaction with gentle shaking for 2 h at room temperature.
   5. After 2 h, remove the tubes from the shaker and add 1.5 µL of triethylamine (TEA) to the reaction in a fume hood.
      NOTE: Triethylamine raises the pH of the solution which stops the aniline tagging reaction and stabilizes the compounds.
      CAUTION: Triethylamine is toxic and causes irritation of the eyes and respiratory tract.
   6. Centrifuge at 13,500 x g for 3 min.
2. Labeling internal standards with ¹³C-aniline solution
   1. Dilute internal stock solution to 80 µM with a final volume of 50 µL.
      NOTE: Concentration of internal standards can be adjusted to levels close to the experimental sample.
   2. Add 5 µL of 200 mg/mL EDC solution.
   3. Add 5 µL of ¹³C-aniline solution.
   4. Vortex the reaction with gentle shaking for 2 h at room temperature.
   5. After 2 h, remove the tubes from the shaker and add 1.5 µL of TEA to the reaction in a fume hood.
   6. Centrifuge at 13,500 x g for 3 min.
3. Combining tagged internal standard and tagged sample
   1. Mix 25 µL of ¹²C-aniline labeled sample with 25 µL of ¹³C-aniline labeled standard.
   2. Transfer to an auto-sampler vial and analyze by the LC/MS procedure.
4. Creating a standard curve for untagged metabolites
   1. Dilute stock solution of untagged metabolites (NAD, NADP, FAD, ACA, and Gly3P) to final concentrations of 320 µM, 80 µM, 20 µM and 5 µM with a volume of 50 µL.
   2. Add 5 µL of 200 mg/mL EDC solution.
   3. Add 5 µL of ¹²C-aniline solution.
   4. Vortex the reaction with gentle shaking for 2 h at room temperature.
   5. After 2 h, remove the tubes from the shaker and add 1.5 µL of TEA to the reaction in a fume hood.
   6. Centrifuge at 13,500 x g for 3 min.
   7. Transfer supernatant to an auto-sampler vial and analyze by the LC/MS procedure.
      NOTE: The untagged metabolites follow the same procedure as the sample to replicate the sample matrix in order to maintain similar ionization efficiency.

5. Setup of LC/MS procedure

1. Preparation of solvents
   1. Prepare 5 mM tri-butylamine (TBA) aqueous solution adjusted to pH 4.75 with acetic acid.
      NOTE: TBA in the mobile phase helps the analytes achieve good resolution and separation¹⁴.
   2. Prepare 5 mM TBA in acetonitrile (ACN).
   3. Prepare wash solvent with 5% water and 95% ACN.
   4. Prepare purge solvent with 95% water and 5% ACN.
2. Setup of MS conditions
   1. Set the mass spectrometer to negative ion mode with a probe temperature of 520 °C, negative capillary voltage of -0.8 kV, positive capillary voltage of 0.8 kV, and set the software to acquire data at 5 points/s.
   2. Set selected ion recordings (SIR) for each metabolite with specified cone voltages and mass over charge (m/z) values. See Table 1.
3. Initializing LC/MS according to manufacturer’s instructions
   1. Prime solvent lines in the solvent manager for 3 min.
   2. Prime wash solvent (5% water, 95% ACN) and purge solvent (95% water, 5% ACN) for 15 s for 5 cycles.
   3. Set the sample manager to 10 °C.
   4. Install a C18 (1.7µm, 2.1mm x 150mm) column and initialize column with 100% ACN at 0.3 mL/min for 10 min.
   5. Condition the column at 95% water and 5% ACN at 0.3 mL/min for 10 min prior to introducing solvents with buffers.
   6. Condition the column at 95% solvent A (5mM TBA aqueous, pH 4.75) and 5% solvent B (5 mM TBA in ACN) at 0.3 mL/min for 10 min.
   7. Set up a gradient protocol with the elution starting at 95% solvent A and 5% solvent B, raised to 70% solvent B in 10 min, raised to 100% solvent B in 2 min and held at 100% solvent B for 3 min. Return to initial conditions (95% solvent A, 5% solvent B) over 1 min and hold for 9 min to re-equilibrate the column.
   8. Condition the column with the gradient protocol 3 times prior to any injections onto the column.
4. Injecting sample and standards
   1. Inject 5 µL of the sample into the column and acquire the appropriate m/z ion intensities for the ¹²C-aniline tagged sample.
   2. Inject 5 µL of the same sample again, but this time acquire the m/z ion intensities for the ¹³C-aniline tagged standards.
      NOTE: Our LC/MS system is unable to acquire both ¹²C and ¹³C m/z intensities at the specified SIR time windows, since it is too much data to acquire in the specified time window. Therefore, we inject the same sample twice.
   3. Inject untagged metabolite standards from lowest concentration to highest and record the appropriate m/z ion intensities.

6. Quantification

1. Creating Export method
   1. In data acquisition software, select File > New Method > Export Method.
   2. Specify a Filename, such as AnilineTagging_Date.
   3. Check the Export ASCII File and choose a directory to export the text file to.
   4. In Report Type, select Summary by All.
   5. In Delimiters, for Column select a ,. For Row, select [cr][if].
   6. In Table, select Export and then Edit Table to include SampleName, Area, Height, Amount and Units.
   7. Save export method.
2. Quantifying metabolites with internal standards using data acquisition software
   1. Under the Sample Sets tab, right click the corresponding LC/MS run and select View as > Channels.
   2. Select all SIR channels for the ¹³C-aniline internal standards of one injection, right click and select Review. 
   3. If the LC Processing Method Layout window does not automatically appear, go to View > Processing Method Layout.
   4. In Processing Method Layout, go to the Integration tab and set ApexTrack as the algorithm.
   5. Go to the Smoothing tab and set the type to Mean and the smoothing level to 13.
      NOTE: Any smoothing level can be selected, as long as it is consistent across all samples.
   6. In the MS Channel tab, disable MS 3D Processing.
   7. In the SIR channel window, integrate each peak, one channel at a time. Once a peak is integrated, go to Options > Fill from Result and the details of the peak will be filled in the Components tab. Change the peak name to the corresponding compound name.
   8. Once all the SIR channels have been evaluated, save the processing method and close window.
   9. Select all SIR channels of the ¹³C-aniline and ¹²C-aniline tagged sample, right click and select Process.
   10. Check the Process box, select Use specified processing method, and choose the processing method that is just saved. Also check the Export box, select Use specified export method and choose the saved export method created earlier. Click OK.
   11. Open the exported text file with Excel and calculate the concentration of the unknown compound using:
       
       where C_x,i is the concentration of the unknown sample for metabolite i, A_x,i is the integrated area of the unknown metabolite i, A_std,i is the integrated area of the internal standard of metabolite i, C_std,i is the concentration of the internal standard of metabolite i, and D is the dilution factor.
3. Quantifying untagged metabolites with standard curve
   1. Under the Sample Sets tab, right click the corresponding LC/MS run and select View as > Channels.
   2. Select all SIR channels for the untagged standards of one injection, right click and select Review. 
   3. If the LC Processing Method Layout window does not automatically appear, go to View > Processing Method Layout.
   4. In Processing Method Layout, go to the Integration tab and set ApexTrack as the algorithm.
   5. Go to the Smoothing tab and set the type to Mean and the smoothing level to 13.
      NOTE: Any smoothing level can be selected, as long as it is consistent across all samples.
   6. In the MS Channel tab, disable MS 3D Processing.
   7. In the SIR channel window, integrate each peak, one channel at a time. Once a peak is integrated, go to Options > Fill from Result and the details of the peak will be filled in the Components tab. Change the peak name to the corresponding compound name.
   8. Once all the SIR channels have been evaluated, save the processing method and close window.
   9. Under the Sample Sets tab, right click on the sample set and select Alter Sample.
   10. Select Amount in the new window.
   11. Select copy from Process method and choose the process method that was just saved.
   12. Enter the concentration of each metabolite for each vial and enter the unit as <μM for each component (or the corresponding unit) and select OK.
   13. Select the sample set again, right click, View as > Channels.
   14. Select all SIR channels of the untagged metabolites for the standards, right click and select Process.
   15. Check the Process box and choose Use specified processing method. Select the appropriate processing method and click OK.
   16. Select SIR channels for all untagged metabolites for the samples, right click and select Process.
   17. Check the Process box, select Use specified processing method, and choose the processing method that was just saved. Also check the Export box, select Use specified export method and choose the saved export method created earlier. Click OK.
   18. Quantify the untagged metabolites with the standard curve and export the results to a text file to the directory specified.

Results

As a proof-of-concept, we used the protocol to quantify metabolites in an E. coli based CFPS system expressing green fluorescent protein (GFP).  The CFPS reaction (14 μL) was quenched and deproteinized with ethanol. The CFPS sample was then tagged with ¹²C-aniline, while standards were tagged with ¹³C-aniline. The tagged sample and standards were then combined and injected into the LC/MS (Figure 1). The protocol detected and quantified 40 metabolites involved i...

Discussion

Cell-free systems have no cell wall, thus there is direct access to metabolites and the biosynthetic machinery without the need for complex sample preparation. However, very little work has been done to develop thorough and robust protocols to quantitatively interrogate cell-free reaction systems. In this study, we developed a fast, robust method to quantify metabolites in cell-free reaction mixtures and potentially in whole-cell extracts. Individual quantification of metabolites in complex mixtures, such as those found ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12C Aniline	Sigma-Aldrich	242284	Aniline 12C
13C labeled aniline	Sigma-Aldrich	485797	Aniline 13C6
3-Phosphoglyceric acid	Sigma-Aldrich	P8877	3PG
Acetic Acid	FisherScientific	AC222140010	ACE
Acetonitrile, LCMS	JT BAKER	9829-03	ACN
Acetyl-coenzyme A	Sigma-Aldrich	A2056	ACA
Acquity UPLC BEH C18 1.7 μM, 2.1 x 150 mm Column	Waters	186002353	Column
Adenosine diphosphate	Sigma-Aldrich	A2754	ADP
Adenosine monophosphate	Sigma-Aldrich	A1752	AMP
Adenosine triphosphate	Sigma-Aldrich	A2383	ATP
Alpha-ketoglutarate	Sigma-Aldrich	K1128	aKG
Citrate	Sigma-Aldrich	251275	CIT
Cytidine diphosphate	Sigma-Aldrich	C9755	CDP
Cytidine monophosphate	Sigma-Aldrich	C1006	CMP
Cytidine triphosphate	Sigma-Aldrich	C9274	CTP
D-glyceraldehyde 3-phosphate	Sigma-Aldrich	39705	GAP
Erythrose 4-phosphate	Sigma-Aldrich	E0377	E4P
Ethanol	Sigma-Aldrich	EX0276	EtOH
Fisher Scientific accuSpin Micro 17 Centrifuge	FisherScientific		Centrifuge
Flavin adenine dinucleotide	Sigma-Aldrich	F6625	FAD
Fructose 1,6-bisphosphate	Sigma-Aldrich	F6803	F16P
Fructose 6-phosphate	Sigma-Aldrich	F3627	F6P
Fumarate	Sigma-Aldrich	F8509	FUM
Gluconate 6-phosphate	Sigma-Aldrich	P7877	6PG
Glucose	Sigma-Aldrich	G8270	GLC
Glucose 6-phosphate	Sigma-Aldrich	G7879	G6P
Glycerol 3-phosphate	Sigma-Aldrich	G7886	Gly3P
Guanosine diphosphate	Sigma-Aldrich	G7127	GDP
Guanosine monophosphate	Sigma-Aldrich	G8377	GMP
Guanosine triphosphate	Sigma-Aldrich	G8877	GTP
Hydrochloric acid	Sigma-Aldrich	258148	HCl
Isocitrate	Sigma-Aldrich	I1252	ICIT
Lactate	Sigma-Aldrich	L1750	LAC
Malate	Sigma-Aldrich	02288	MAL
myTXTL - Sigma 70 Master Mix Kit	ArborBiosciences	507024	Cell-free protein synthesis
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride	Sigma-Aldrich	03449	EDC
Nicotinamide adenine dinucleotide	Sigma-Aldrich	43410	NAD
Nicotinamide adenine dinucleotide phosphate	Sigma-Aldrich	N5755	NADP
Nicotinamide adenine dinucleotide phosphate reduced	Sigma-Aldrich	481973	NADPH
Nicotinamide adenine dinucleotide reduced	Sigma-Aldrich	N8129	NADH
Oxalacetate	Sigma-Aldrich	O4126	OAA
Phosphoenolpyruvate	Sigma-Aldrich	P0564	PEP
Pyruvate	Sigma-Aldrich	P5280	PYR
Ribose 5-phosphate	Sigma-Aldrich	R7750	R5P
Ribulose 5-phosphate	CarboSynth	MR45852	RL5P
Sedoheptulose 7-phosphate	CarboSynth	MS07457	S7P
Succinate	Sigma-Aldrich	S3674	SUCC
Tributylamine	Sigma-Aldrich	90780	TBA
Triethylamine	FisherScientific	O4884	TEA
ultrapure water	FisherScientific	10977-015	water
Uridine diphosphate	Sigma-Aldrich	U4125	UDP
Uridine monophosphate	Sigma-Aldrich	U6375	UMP
Uridine triphosphate	Sigma-Aldrich	U6625	UTP
VWR Heavy Duty Vortex	VWR		Vortex
Water, LCMS	JT BAKER	9831-03	WATER
Waters Acquity H UPLC Class Quaternary Solvent Manager	Waters		LCMS
Waters Acquity H UPLC Class Sample Manager FTN	Waters		LCMS
Waters Acquity Qda detector	Waters		LCMS
Waters Empower 3	Waters		Software
Waters LCMS Total Recovery Vial	Waters	186000384c	LCMS Vial