Profile of native N-linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry

Caroline S. Chu, Milady R. Niñonuevo, Brian H. Clowers, Patrick D. Perkins, Hyun Joo An, Hongfeng Yin, Kevin Killeen, Suzanne Miyamoto, Rudolf Grimm, Carlito B Lebrilla

Research output: Contribution to journalArticle

119 Scopus citations

Abstract

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N-linked oligosaccharides released from human serum without derivatization has been developed using on-line nanoLC and high resolution TOF MS. The N-linked oligosaccharides were analyzed with MALDI FT-ICR MS and microchip LC MS (HPLC-Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 4360.075 mm2 i.d. analytical column) and the high capacity chip (160 nL enrichment column, 14060.075 mm2 i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N-linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for ~ 96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ~ 4%.

Original languageEnglish (US)
Pages (from-to)1939-1951
Number of pages13
JournalProteomics
Volume9
Issue number7
DOIs
StatePublished - Apr 2009

Keywords

  • FT-ICR MS
  • HPLC/Chip-TOF MS
  • Human serum
  • N-linked
  • Oligosaccharides

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

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