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Peer-reviewedCC BY-NC-ND 4.0Analytical Chemistry

Automated Quenching and Extraction for High-Precision Metabolomics Uncovers Distinct Metabolome Trajectories across Escherichia coli Lineages

  • Kenya Tanaka1,2,3
  • Takahiro Bamba2
  • Musashi Takenaka2
  • Yoshimi Hori1
  • Takanobu Yoshida1
  • Mami Matsuda1
  • Akihiko Kondo1,2,4,5
  • Yoshihiro Hayakawa6
  • Tomohisa Hasunuma1,2,4,5
6 affiliations
  1. 1Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
  2. 2Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
  3. 3Research Center for Solar Energy Chemistry, Graduate School of Engineering Science, The University of Osaka, Toyonaka, Osaka 560-8531, Japan
  4. 4RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan
  5. 5Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
  6. 6Solutions Center of Excellence, Analytical & Measuring Instruments Division, Shimadzu Corporation, 1, Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan
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Abstract

Quantitative microbial metabolomics requires rapid quenching and highly standardized extraction, yet these pre-analytical steps remain labor-intensive and difficult to reproduce, limiting time-resolved intracellular measurements under controlled bioreactor cultivation. Here, we present an integrated robotic platform that couples parallel fermenter operation with circulation-based sampling, immediate cold-alcohol quenching, automated refrigerated centrifugation, interface-aware supernatant removal, and solvent extraction with controlled heating and mixing, followed by plate-based storage for liquid chromatography–tandem mass spectrometry (LC–MS/MS). To benchmark the precision of the automated preparation module, we compared repeated LC–MS/MS measurements of a single extract with measurements of independently processed samples subjected to post-quench automated harvesting, extraction, and transfer. The analysis was restricted to 76 analytes consistently detected in all replicates of both datasets. The mean coefficients of variation increased from 6.92% for repeated LC–MS/MS measurements to 8.82% for automated preparation, corresponding to a modest increase of 1.90 percentage points. These results indicate that post-quench automated preparation introduces a small but measurable additional source of variability while maintaining mean analytical variation below 10%. We then performed time-course endo- and exometabolomics across four widely used Escherichia coli lineages (BW25113, BL21­(DE3), W, and C). Principal component analysis resolved strain-specific trajectories throughout batch growth and revealed strain-specific metabolic phenotypes, including coordinated accumulation of sugar phosphates and 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway intermediates in strain C, and comparatively high intracellular pools of branched-chain amino acids in strain W. This platform enables standardized and traceable automated preparation for dense, time-resolved metabolomics and provides directly measurable phenotypes for host selection and metabolic engineering.

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Cite

BibTeXRIS
BibTeX
@article{tanaka2026automated,
  title = {{Automated Quenching and Extraction for High-Precision Metabolomics Uncovers Distinct Metabolome Trajectories across Escherichia coli Lineages}},
  author = {Kenya Tanaka and Takahiro Bamba and Musashi Takenaka and Yoshimi Hori and Takanobu Yoshida and Mami Matsuda and Akihiko Kondo and Yoshihiro Hayakawa and Tomohisa Hasunuma},
  journal = {Analytical Chemistry},
  year = {2026},
  month = oct,
  doi = {10.1021/acs.analchem.6c01820},
  url = {https://doi.org/10.1021/acs.analchem.6c01820},
  note = {CC BY-NC-ND 4.0},
}
RIS
TY  - JOUR
TI  - Automated Quenching and Extraction for High-Precision Metabolomics Uncovers Distinct Metabolome Trajectories across Escherichia coli Lineages
AU  - Kenya Tanaka
AU  - Takahiro Bamba
AU  - Musashi Takenaka
AU  - Yoshimi Hori
AU  - Takanobu Yoshida
AU  - Mami Matsuda
AU  - Akihiko Kondo
AU  - Yoshihiro Hayakawa
AU  - Tomohisa Hasunuma
PY  - 2026
DA  - 2026/10/19
JO  - Analytical Chemistry
DO  - 10.1021/acs.analchem.6c01820
UR  - https://doi.org/10.1021/acs.analchem.6c01820
AB  - Quantitative microbial metabolomics requires rapid quenching and highly standardized extraction, yet these pre-analytical steps remain labor-intensive and difficult to reproduce, limiting time-resolved intracellular measurements under controlled bioreactor cultivation. Here, we present an integrated robotic platform that couples parallel fermenter operation with circulation-based sampling, immediate cold-alcohol quenching, automated refrigerated centrifugation, interface-aware supernatant removal, and solvent extraction with controlled heating and mixing, followed by plate-based storage for liquid chromatography–tandem mass spectrometry (LC–MS/MS). To benchmark the precision of the automated preparation module, we compared repeated LC–MS/MS measurements of a single extract with measurements of independently processed samples subjected to post-quench automated harvesting, extraction, and transfer. The analysis was restricted to 76 analytes consistently detected in all replicates of both datasets. The mean coefficients of variation increased from 6.92% for repeated LC–MS/MS measurements to 8.82% for automated preparation, corresponding to a modest increase of 1.90 percentage points. These results indicate that post-quench automated preparation introduces a small but measurable additional source of variability while maintaining mean analytical variation below 10%. We then performed time-course endo- and exometabolomics across four widely used Escherichia coli lineages (BW25113, BL21­(DE3), W, and C). Principal component analysis resolved strain-specific trajectories throughout batch growth and revealed strain-specific metabolic phenotypes, including coordinated accumulation of sugar phosphates and 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway intermediates in strain C, and comparatively high intracellular pools of branched-chain amino acids in strain W. This platform enables standardized and traceable automated preparation for dense, time-resolved metabolomics and provides directly measurable phenotypes for host selection and metabolic engineering.
SN  - 0003-2700
SN  - 1520-6882
ER  -