High-Throughput Quantitative
Analysis of Total <i>N</i>-Glycans by Matrix-Assisted Laser
Desorption/Ionization
Time-of-Flight Mass Spectrometry
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Abstract
Accurate and reproducible quantification of glycans from
protein
drugs has become an important issue for quality control of therapeutic
proteins in biopharmaceutical and biotechnology industries. Mass spectrometry
is a promising tool for both qualitative and quantitative analysis
of glycans owing to mass accuracy, efficiency, and reproducibility,
but it has been of limited success in quantitative analysis for sialylated
glycans in a high-throughput manner. Here, we present a solid-phase
permethylation-based total <i>N</i>-glycan quantitative
method that includes <i>N</i>-glycan releasing, purification,
and derivatization on a 96-well plate platform. The solid-phase neutralization
enabled us to perform reliable absolute quantification of the acidic <i>N</i>-glycans as well as neutral <i>N</i>-glycans
from model glycoproteins (i.e., chicken ovalbumin and porcine thyroglobulin)
by only using matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS). Furthermore, low-abundance sialylated <i>N</i>-glycans from human serum prostate specific antigen (PSA),
an extremely valuable prostate cancer marker, were initially quantified,
and their chemical compositions were proposed. Taken together, these
results demonstrate that our all-inclusive glycan preparation method
based on a 96-well plate platform may contribute to the precise and
reliable qualitative and quantitative analysis of glycans