Absolute quantification of choline-related biomarkers in breast cancer biopsies by liquid chromatography electrospray ionization mass spectrometry

Maria Chiara Mimmi, Nicoletta Finato, Gloria Pizzolato, Carlo Alberto Beltrami, Federico Fogolari, Alessandra Corazza, Gennaro Esposito

Research output: Contribution to journalArticle

Abstract

It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis. The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many 'targeted' approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Here we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis. The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio.

Original languageEnglish (US)
Pages (from-to)71-83
Number of pages13
JournalAnalytical Cellular Pathology
Volume36
Issue number3-4
DOIs
StatePublished - Nov 25 2013

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Electrospray Ionization Mass Spectrometry
Choline
Liquid Chromatography
Phosphorylcholine
Biomarkers
Breast Neoplasms
Biopsy
Protons
Metabolomics
Metabolome
Reverse-Phase Chromatography
Tumor Biomarkers
Hydrophobic and Hydrophilic Interactions
Amides
Calibration
Chromatography
Neoplasms
Magnetic Resonance Spectroscopy
Cell Culture Techniques

Keywords

  • $^1$H-NMR
  • breast cancer
  • choline phospholipid metabolism
  • hydrophilic interaction chromatography
  • LC-ESI-MS
  • Metabolomics

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Medicine
  • Pathology and Forensic Medicine
  • Cell Biology

Cite this

Absolute quantification of choline-related biomarkers in breast cancer biopsies by liquid chromatography electrospray ionization mass spectrometry. / Mimmi, Maria Chiara; Finato, Nicoletta; Pizzolato, Gloria; Beltrami, Carlo Alberto; Fogolari, Federico; Corazza, Alessandra; Esposito, Gennaro.

In: Analytical Cellular Pathology, Vol. 36, No. 3-4, 25.11.2013, p. 71-83.

Research output: Contribution to journalArticle

Mimmi, Maria Chiara ; Finato, Nicoletta ; Pizzolato, Gloria ; Beltrami, Carlo Alberto ; Fogolari, Federico ; Corazza, Alessandra ; Esposito, Gennaro. / Absolute quantification of choline-related biomarkers in breast cancer biopsies by liquid chromatography electrospray ionization mass spectrometry. In: Analytical Cellular Pathology. 2013 ; Vol. 36, No. 3-4. pp. 71-83.
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