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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
Author(s) -
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.
Publication year - 2009
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200800249
Subject(s) - chromatography , mass spectrometry , microfluidics , glycan , time of flight mass spectrometry , chemistry , microfluidic chip , chip , analytical chemistry (journal) , materials science , nanotechnology , computer science , glycoprotein , biochemistry , ion , organic chemistry , ionization , telecommunications
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, 43×0.075 mm 2 i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140×0.075 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 ∼96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ∼4%.