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 ....

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-....

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