Untargeted Metabolic Profiling of Oral Cancer Cells Using Capillary Ion Chromatography Coupled with an Orbitrap Mass Spectrometer

Importance of the Topic

Untargeted profiling of polar anionic metabolites provides a direct snapshot of cellular function and is critical for identifying biomarkers in cancer research. Metabolomics complements genomics and proteomics by capturing end‐product changes in metabolic pathways that reflect disease progression and response to therapy.

Objectives and Study Overview

This study compares three chromatographic approaches—capillary ion chromatography (IC), hydrophilic interaction liquid chromatography (HILIC), and reversed‐phase ultra-high-pressure LC (RP-UHPLC)—coupled to a high-resolution Orbitrap mass spectrometer for untargeted metabolite profiling of oral squamous cell carcinoma (OSCC) cell lines, including cancer stem‐like cells (CSCs) and non‐stem controls.

Methodology and Instrumentation

Cells from UMSCC1, UMSCC5, and CSC lines were snap-frozen in liquid nitrogen and extracted with methanol/water. Three separation methods were evaluated:

• Capillary IC with KOH gradient eluent and electrolytic suppression to pure water, 25 µL/min (plus 10 µL/min makeup flow)
• HILIC using a ZIC-HILIC column at 250 µL/min
• RP-UHPLC on a C8 column at 450 µL/min

Negative-ion electrospray Q Exactive Orbitrap MS provided full-scan (67–1000 m/z, 70 000 resolution) and data‐dependent MS/MS spectra. Data processing involved SIEVE software, KEGG and METLIN database searches, and XCMS/metaXCMS for meta-analysis.

Used Instruments

• Dionex ICS-4000 Capillary HPIC System with EGC-KOH cartridge, IC Cube module, ACES 300 suppressor, and conductivity detector
• Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer
• Thermo Scientific UltiMate 3000 RSLC HPG System for HILIC and RP-UHPLC

Main Results and Discussion

Capillary IC-Orbitrap MS achieved femtomole detection limits (0.05–0.5 nmol/L) for 21 key polar metabolites, surpassing HILIC by 10–100× and RP-UHPLC. Baseline resolution was obtained for isomeric sugar phosphates and tricarboxylic acid cycle intermediates, with inter-day retention time and intensity RSDs <8%. In OSCC cell lysates, capillary IC detected 65 features versus 38 by HILIC and 29 by RP-UHPLC. Pathway mapping and pairwise comparisons revealed significant alterations in glycolysis and TCA cycle upon SOX11 knockdown, and a second-order meta-analysis across three cell models identified 218 common differential metabolites.

Benefits and Practical Applications

The IC-Orbitrap approach offers:

• Superior selectivity and sensitivity for charged polar analytes
• Enhanced resolution of isobaric and isomeric metabolites
• Robust untargeted discovery of metabolic biomarkers in complex biological matrices

Future Trends and Opportunities

Advances may include faster IC gradients with improved cartridges, expanded high-resolution MS/MS libraries for better identification of isomers, integration with multi-omics workflows, and broader application to clinical and environmental metabolomics.

Conclusion

Capillary ion chromatography coupled to high-resolution Orbitrap mass spectrometry provides unmatched performance for untargeted profiling of polar anionic metabolites in oral cancer cells, outperforming HILIC and RP-UHPLC methods in both sensitivity and chromatographic resolution. This platform enables comprehensive pathway analysis and biomarker discovery in metabolomics studies.

Reference

1. Cubbon S, Antonio C, Wilson JD, Thomas-Oates J. Metabolomic applications of HILIC–LC–HRAM. Mass Spectrom Rev. 2010;29(4):671–684.
2. Creek DJ, Jankevics A, Breitling R, Watson DG, Barrett MP, Burgess KEV. Toward global metabolomics analysis with HILIC–LC–HRAM. Anal Chem. 2011;83:8703–8710.
3. DeFrancesco L. Capillary electrophoresis: Finding a niche. Today’s Chemist at Work. 2002 Feb.
4. Cruces-Blanco C, Gamiz-Gracia L, Garcia-Campana AM. Applications of capillary electrophoresis in forensic analytical chemistry. Trends Anal Chem. 2007;26(3):216.
5. Gika HG, Theodoridis GA, Plumb RS, Wilson ID. Current practice of LC-MS in metabolomics and metabonomics. Pharm Biomed Anal. 2014;87:12–25.
6. Theodoridis GA, Gika HG, Want EJ, Wilson ID. LC-MS based global metabolite profiling: A review. Anal Chim Acta. 2012;711:7–16.
7. Bruggink C, Maurer R, Herrmann H, Cavalli S, Hoefler F. Analysis of carbohydrates by anion exchange chromatography and MS. J Chromatogr A. 2005;1085:104–109.
8. Burgess K, Creek D, Dewsbury P, Cook K, Barrett MP. Semi-targeted metabolite analysis using capillary-flow IC–HRAM MS. Rapid Commun Mass Spectrom. 2011;25:3447–3452.
9. Wang J, Christison TT, Misuno K, et al. Metabolomic profiling of anionic metabolites in head and neck cancer cells by capillary ion chromatography with Orbitrap MS. Anal Chem. 2014;86(10):5116–5124.
10. Oral Cancer Facts. Oral Cancer Foundation; 2014 Aug.
11. GBD 2013 Mortality and Causes of Death Collaborators. Global mortality for 240 causes of death: a systematic analysis for GBD 2013. Lancet. 2015;385(9963):117–171.
12. SEER Stat Fact Sheets: Oral Cavity and Pharynx Cancer. NCI SEER Program; accessed Mar 2015.
13. Yonezawa K, Nishiumi S, Kitamoto-Matsuda J, et al. Serum and tissue metabolomics of head and neck cancer. Cancer Genomics Proteomics. 2013;10:233–238.
14. Ye G, Liu Y, Yin P, et al. Study of induction chemotherapy efficacy in oral carcinoma using pseudotargeted metabolomics. J Proteome Res. 2014;13:1994–2004.
15. Misuno K, Liu X, Feng S, Hu S. Quantitative proteomic analysis of sphere-forming stem-like oral cancer cells. Stem Cell Res Ther. 2013;4:156.
16. Lorenz MA, Burant CF, Kennedy RT. Reducing time and increasing sensitivity in sample prep for adherent mammalian cell metabolomics. Anal Chem. 2011;83:3406–3414.
17. Wang J, Christison T, Backiel K, Ji G, Hu S, Lopez L, Huang Y, Huhmer A. Pathway-targeted metabolomic analysis using IC-MS. Thermo Fisher Scientific AN64317-EN; 2015.
18. Hu S, Wang J, Christison T, Lopez L, Huang Y. Targeted metabolomic analysis of head and neck cancer cells using IC–Q Exactive HF MS. Anal Chem. 2015;Epub May 22.
19. Soga T, Ueno Y, Naraoka H, Ohashi Y, Tomita M, Nishioka T. Simultaneous determination of anionic intermediates by CE–ESI-MS. Anal Chem. 2002;74:2233–2239.