Coupling flash liquid chromatography with mass spectrometry for enrichment and isolation of milk oligosaccharides for functional studies

John S. Strum, Danielle Aldredge, Daniela Barile, Carlito B Lebrilla

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories. The online mass detector enabled real-time optimization of chromatographic parameters to favor separation of oligosaccharides that would otherwise be indistinguishable from coeluting components with a nonspecific detector. Unlike previously described preparative LC/MS techniques, we have employed a dynamic flow connection that permits any flow rate from the flash system to be delivered from 1 to 200 ml/min without affecting the ionization conditions of the mass spectrometer. A new way of packing large amounts of graphitized carbon allowed the enrichment and separation of milligram quantities of structurally heterogeneous mixtures of human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). Abundant saccharide components in milk, such as lactose and lacto-N-tetraose, were separated from the rarer and less abundant oligosaccharides that have greater structural diversity and biological functionality. Neutral and acidic HMOs and BMOs were largely separated and enriched with a dual binary solvent system.

Original languageEnglish (US)
Pages (from-to)87-96
Number of pages10
JournalAnalytical Biochemistry
Volume424
Issue number2
DOIs
StatePublished - May 15 2012

Keywords

  • Bovine milk oligosaccharides
  • Flash chromatography
  • Fraction collection
  • Graphitized carbon
  • Human milk oligosaccharides
  • Low-pressure liquid chromatography
  • Mass spectrometry
  • Online detection
  • Size exclusion

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

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