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

Proteomics. 2009 Apr;9(7):1939-51. doi: 10.1002/pmic.200800249.


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, 43 x 0.075 mm(2) i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140 x 0.075 mm(2) 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 approximately 96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining approximately 4%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Proteins / chemistry
  • Blood Proteins / metabolism*
  • Chromatography, High Pressure Liquid*
  • Glycosylation
  • Humans
  • Mass Spectrometry*
  • Microchip Analytical Procedures
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
  • Polysaccharides* / chemistry
  • Polysaccharides* / metabolism
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Blood Proteins
  • Polysaccharides
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase