Integrated LC/MS Workflow for the Analysis
of Labeled and Native N-Glycans from
Proteins Using a Novel Mixed-Mode Column
and a Q Exactive Mass Spectrometer
Udayanath Aich
1
, Julian Saba
2
, Rosa Viner
2
, Xiaodong Liu
1
, Srinivasa Rao
1
, Yury Agroskin
1
, Andreas Huhmer
2
and Chris Pohl
1
1
Thermo Fisher Scientific, Sunnyvale, CA;
2
Thermo Fisher Scientific, San Jose, CA
Application Note 595
Key Words
GlycanPac AXH-1, HILIC, WAX, glycomics, glycoproteins, glycopeptides,
glycans, labeled
N
-glycans, Q Exactive, SimGlycan software
Goal
Develop a comprehensive method for the structural characterization of
released glycans from proteins. The described integrated method covers
sample preparation, separation, mass spectrometry data acquisition, and
analysis.
Introduction
Glycans are widely distributed in biological systems in
‘free state’ as well as conjugated forms such as
glycoproteins, glycolipids, and proteoglycans. They play
significant roles in many biological and physiological
processes, including recognition and regulatory functions,
cellular communication, gene expression, cellular
immunity, growth, and development.
1
Glycans can affect
efficacy and safety of protein based drugs. For example,
recombinant proteins and monoclonal antibodies (mAb)
are often dependent on the structure and types of glycans
attached to the proteins.
2
The structures of glycans are
diverse, complex, and heterogeneous due to post-
translational modifications (PTMs) and physiological
conditions. Minor changes in glycan structure can result
in striking differences in biological functions and clinical
applications. The structural characterization of glycans is
essential in bio-therapeutics and bio-pharmaceutical
projects.
3
In addition to the characterization of the sugar
sequence, the analysis must elucidate linkages and
separate all isomeric, charge, and branching variations
of glycans.
Liquid chromatography (LC) coupled to mass
spectrometry (MS) has emerged as one of the most
powerful tools for the structural characterization of
glycans. Hydrophilic interaction liquid chromatography
(HILIC) columns based on amide, amine, or zwitterionic-
based packing materials are often used for glycan analysis.
These HILIC columns separate glycans mainly by
hydrogen bonding, resulting in size and composition-
based separation. A limitation of this approach is that
identification of the glycan charge state is not possible due
to the fact that glycans of different charge states are
intermingled in the separation envelope.
The Thermo Scientific
™
GlycanPac
™
AXH-1 column is a
high-performance HPLC/UHPLC column specifically
designed for structural analysis of glycans, either labeled
or native, by LC-fluorescence or LC/MS methods.The
GlycanPac AXH-1 column is based on innovative
mixed-mode surface chemistry combining both weak
anion-exchange (WAX) and HILIC retention mechanisms.
The WAX functionality provides retention and selectivity
for negatively charged glycans, while the HILIC mode
facilitates the separation of glycans according to their
charge, polarity, and size. As a result, the GlycanPac
AXH-1 column provides unparalleled separation
capabilities for glycans.
LC-MS/MS analysis of glycans requires the processing of
large sets of data. The incorporation of SimGlycan
®
software (PREMIER Biosoft) alleviates this issue, thus
enabling the development of a true high-throughput
workflow.
This application note presents a step-by-step method for
the release, labeling, separation, and structural elucidation
of
N
-glycans from proteins by LC-MS/MS.
