TY - JOUR
T1 - Chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated N-linked glycans
T2 - A potential methodology for cancer-biomarker discovery
AU - Alley, William R.
AU - Madera, Milan
AU - Mechref, Yehia
AU - Novotny, Milos V.
N1 - Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 2010/6/15
Y1 - 2010/6/15
N2 - The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Quantification of glycomic-profile changes is becoming increasingly important in the search for disease biomarkers. Here, we report a highly reproducible combination of a glycomic sample preparation/solid-phase derivatization of glycoprotein-derived N-linked glycans with their subsequent microchip-based separation and mass-spectrometric (MS) measurements. Following our previously described reductive β-elimination for O-linked glycans with ammonia-borane complex to reduce N-linked structures, the N-linked alditol structures are effectively methylated in dimethylformamide medium to avoid artefacts in MS measurements. Reversed-phase microfluidic liquid chromatography (LC) of methylated N-linked oligosaccharide alditols resolved some closely related structures into regular retention increments, aiding in their structural assignments. Optimized LC gradients, together with nanospray MS, have been applied here in the quantitative measurements of N-linked glycans in blood serum, distinguishing breast cancer patients from control individuals.
AB - The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Quantification of glycomic-profile changes is becoming increasingly important in the search for disease biomarkers. Here, we report a highly reproducible combination of a glycomic sample preparation/solid-phase derivatization of glycoprotein-derived N-linked glycans with their subsequent microchip-based separation and mass-spectrometric (MS) measurements. Following our previously described reductive β-elimination for O-linked glycans with ammonia-borane complex to reduce N-linked structures, the N-linked alditol structures are effectively methylated in dimethylformamide medium to avoid artefacts in MS measurements. Reversed-phase microfluidic liquid chromatography (LC) of methylated N-linked oligosaccharide alditols resolved some closely related structures into regular retention increments, aiding in their structural assignments. Optimized LC gradients, together with nanospray MS, have been applied here in the quantitative measurements of N-linked glycans in blood serum, distinguishing breast cancer patients from control individuals.
UR - http://www.scopus.com/inward/record.url?scp=77953555196&partnerID=8YFLogxK
U2 - 10.1021/ac100131e
DO - 10.1021/ac100131e
M3 - Article
C2 - 20491449
AN - SCOPUS:77953555196
VL - 82
SP - 5095
EP - 5106
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 12
ER -