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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

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...

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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
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cited_by cdi_FETCH-LOGICAL-c5899-93170293323f91328d8194a0404be82d3fe2bcb81735d31831cc14d5bb7f30943
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creator Chu, Caroline S
Niñonuevo, Milady R
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Lebrilla, Carlito B
description 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%.
doi_str_mv 10.1002/pmic.200800249
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source Wiley:Jisc Collections:Wiley Read and Publish Open Access 2024-2025 (reading list)
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
title 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
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