Bacterial Metabolomics: Sample Preparation Methods

Table 1

Summary of optimized quenching methods in bacterial metabolomics studies.

Bacteria	Approach	Optimal quenching method	Findings	Ref.
Gram-negative
Saccharophagus degradans	Untargeted	(−70°C, 70%) methanol	(i) Severe cell leakage induced significant loss of intracellular metabolites.	[90]
E. coli	Untargeted	(−48°C, 60%) methanol	(i) Produce the highest recovery of intracellular metabolites with highest peak of metabolites detected.	[71]
E. coli	Untargeted	Automated fast filtration with on-filter (−45°C, 60%) methanol	(i) Significantly higher intracellular concentrations of amino acids were obtained.	[44]
			(ii) Minimize total sampling time and metabolite leakage.
E. coli	Untargeted	(−50°C) 60% methanol/40% glycerol	(i) Methanol/glycerol significantly reduced leakage of ATP (15–16%) compared to 60% methanol.	[92]
E. coli	Untargeted	Liquid nitrogen	(i) Enhanced metabolites recovery compared to 60% methanol (−40°C) that caused more metabolites leakage.	[24]
Gram-positive
B. licheniformis	Untargeted	(−40°C) 60% methanol/0.9% NH4HCO3	(i) Detection of 127 metabolites with vast amount of amino acids, organic acids, and carbohydrates.	[87]
			(ii) Improved protein exudation and reduced metabolites leakage.
			(iii) NH4HCO3 is suitable for LCMS requirements of metabolomics analysis.
C. crescentus	Untargeted	(−20°C, 80%) methanol	(i) Higher recovery of polar compound including CoA and CoA thioester derivatives, citric acid, and some nucleotides.	[59]
P. fluorescens	Untargeted	Cold glycerol-saline (3 : 2), glycerol-water (3 : 2), glycerol-mannitol (3 : 2)	(i) Glycerol-saline (−23°C) produced higher detection and less metabolite leakage compared to cold methanol.	[85]
L. bulgaricus	Untargeted	(−20°C, 80%) methanol: glycerol	(i) The solvent applicable to other Gram-positive bacteria (S. coelicolor) and yeast (S. cerevisiae).	[91]
MRSA	Untargeted	On-filter culture (20°C, 60%) ethanol	(i) No significant metabolic disruption.	[36]
			(ii) Good reproducibility and consistency.
S. aureus	Untargeted	Fast filtration followed by (−20°C, 0%) ethanol and liquid nitrogen	(i) Separation of cells prior to quenching caused no significant metabolites leakage with better energy charge.	[49]
			(ii) Effective quenching is achieved by (−20°C, 60%) ethanol.
B. subtilis	Untargeted	Liquid nitrogen with vacuum filtration	(i) Improved metabolic arrest during filtration.	[56]
C. glutamicum, E. coli	Untargeted	(−20°C, 40%) ethanol and 0.8% (w/v) sodium chloride	(i) Highest detection and identification of metabolites with ethanol quenching (118 metabolites) compared to 60% methanol (−50°C) and glycerol-saline (−20°C).	[83]
Lactobacillus plantarum	Targeted	(−40°C, 60%) methanol with 0.85% ammonium carbonate	(i) 60% methanol (−40°C), 60% methanol (−40°C)/ 0.85% NaCl/HEPES (70 mM) showed more than 10% cell leakage.	[58]
MRSA	Targeted	Filter-based system with (−20°C) ethanol plus liquid nitrogen	(i) Highest recovery of almost all amino acids.	[82]
			(ii) Reduced metabolites leakage.
Streptomyces ZYJ-6	Targeted	Isoamylol: (acetone: ethanol, 1 : 1) (5 : 1, v/v)	(i) 60% methanol produced the largest metabolite leakage, followed by acetone: base, methanol: base, and propanol: base.	[66]