Analytical and Bioanalytical Chemistry

, Volume 405, Issue 15, pp 5279–5289

Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells

  • Huichang Bi
  • Kristopher W. Krausz
  • Soumen K. Manna
  • Fei Li
  • Caroline H. Johnson
  • Frank J. Gonzalez
Research Paper

DOI: 10.1007/s00216-013-6927-9

Cite this article as:
Bi, H., Krausz, K.W., Manna, S.K. et al. Anal Bioanal Chem (2013) 405: 5279. doi:10.1007/s00216-013-6927-9

Abstract

In this study, a liquid chromatography mass spectrometry (LC/MS)-based metabolomics protocol was optimized for quenching, harvesting, and extraction of metabolites from the human pancreatic cancer cell line Panc-1. Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in water were compared for sample harvesting. Four different extraction methods were compared to investigate the efficiency of intracellular metabolite extraction, including pure acetonitrile, methanol, methanol/chloroform/H2O, and methanol/chloroform/acetonitrile. The separation efficiencies of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) with UPLC-QTOF-MS were also evaluated. Global metabolomics profiles were compared; the number of total detected features and the recovery and relative extraction efficiencies of target metabolites were assessed. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Direct scraping after flash quenching with liquid nitrogen was chosen to harvest Panc-1 cells which allowed for samples to be stored before extraction. Methanol/chloroform/H2O was chosen as the optimal extraction solvent to recover the highest number of intracellular features with the best reproducibility. HILIC had better resolution for intracellular metabolites of Panc-1 cells. This optimized method therefore provides high sensitivity and reproducibility for a variety of cellular metabolites and can be applicable to further LC/MS-based global metabolomics study on Panc-1 cell lines and possibly other cancer cell lines with similar chemical and physical properties.

Figure

Optimized harvesting, extraction and analytical protocols for cell metabolomics analysis.

Keywords

Metabolomics Sample preparation Metabolite extraction Panc-1 cell line HILIC 

Supplementary material

216_2013_6927_MOESM1_ESM.pdf (221 kb)
ESM 1(PDF 220 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Huichang Bi
    • 1
    • 2
  • Kristopher W. Krausz
    • 2
  • Soumen K. Manna
    • 2
  • Fei Li
    • 2
  • Caroline H. Johnson
    • 2
    • 3
  • Frank J. Gonzalez
    • 2
  1. 1.School of Pharmaceutical SciencesSun Yat-sen UniversityGuangzhouChina
  2. 2.Laboratory of Metabolism, Center for Cancer ResearchNational Cancer Institute, NIHBethesdaUSA
  3. 3.Center for Metabolomics and Mass SpectrometryThe Scripps Research InstituteLa JollaUSA