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 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%.
Chu
Caroline S
CS
Department of Chemistry, University of California at Davis, Davis, CA 95616, USA.
Niñonuevo
Milady R
MR
Clowers
Brian H
BH
Perkins
Patrick D
PD
An
Hyun Joo
HJ
Yin
Hongfeng
H
Killeen
Kevin
K
Miyamoto
Suzanne
S
Grimm
Rudolf
R
Lebrilla
Carlito B
CB
D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D046208:Microchip Analytical Procedures; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001798:Blood Proteins; D011134:Polysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase