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

• Published in: Proteomics (Weinheim) 2009-04, Vol.9 (7), p.1939-1951
• Main Authors: Chu, Caroline S, Niñonuevo, Milady R, Clowers, Brian H, Perkins, Patrick D, An, Hyun Joo, Yin, Hongfeng, Killeen, Kevin, Miyamoto, Suzanne, Grimm, Rudolf, Lebrilla, Carlito B
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blood Proteins - chemistry; Blood Proteins - metabolism; Chromatography, High Pressure Liquid; FT‐ICR MS; Fundamental and applied biological sciences. Psychology; Glycosylation; HPLC/Chip‐TOF MS; Human serum; Humans; Mass Spectrometry; Microchip Analytical Procedures; Miscellaneous; N‐linked; Oligosaccharides; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism; Polysaccharides - chemistry; Polysaccharides - metabolism; Proteins; Reproducibility of Results; Sensitivity and Specificity
• ISSN: 1615-9853; 1615-9861
• 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, 43x0.075 mm² i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140x0.075 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%.