Application Note 20786
Structural Analysis of Labeled
N
-Glycans
from Proteins by LC-MS/MS Separated
Using a Novel Mixed-Mode Stationary
Phase
Udayanath Aich
1
, Julian Saba
2
, Xiaodong Liu
1
, Srinivasa Rao
1
, and Chris Pohl
1
1
Thermo Fisher Scientific, Sunnyvale, CA, USA;
2
Thermo Fisher Scientific, Mississuaga, ON, Canada
Introduction
Glycans are involved in a wide range of biological
and physiological processes, including recognition and
regulatory functions, cellular communication, gene
expression, cellular immunity, growth, and
development [1]. Glycans are commonly investigated as
important species in therapeutic protein drug development
because there is strong evidence that bioactivity and
efficacy are affected by glycosylation [2]. Commonly, both
the structure and types of glycans attached to the proteins
are examined. Understanding, measuring, and controlling
glycosylation in glycoprotein-based drugs, glycan content
of glycoprotein products, as well as thorough
characterization of biosimilars has become increasingly
important.
The structures of glycans are highly diverse, complex, and
heterogeneous due to post-translational modifications.
This makes it challenging to comprehensively characterize
glycan profiles and determine their structures [3]. It is
therefore essential to separate all isomeric, charge, and
branching glycan variations to understand the detailed
structure of the glycans by LC-MS/MS methods.
Various HPLC separation modes have been used for the
analysis of glycans, including normal phase (NP) or
hydrophilic interaction (HILIC) chromatography,
ion-exchange (IEX) chromatography, and reversed-phase
(RP) chromatography. Because they are highly
Key Words
GlycanPac AXH-1, LC/MS, LC-MS/MS, HILIC, WAX, mixed-mode, labeled
N
-glycans, UHPLC, MS detection, Q Exactive, charge, SimGlycan software
Abstract
This application note describes the liquid chromatography-mass
spectrometry (LC/MS) analysis of fluorescently labeled
N
-glycans released
from proteins. The chromatographic separation is carried out with a novel
Thermo Scientific™ GlycanPac™ AXH-1 (1.9 µm, 2.1 × 150 mm) column
for high-resolution and high-throughput analysis of glycans. This column
possesses unique selectivity that provides separation of glycans based on
charge, size, and polarity. MS and MS/MS analyses are performed using
a Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap™ mass
spectrometer in negative ion mode to provide detailed structural information
of
N
-glycans released from proteins.
hydrophilic, polar substances, neutral glycans are commonly
separated using amide HILIC columns, such as the Thermo
Scientific™ Accucore™ 150-Amide-HILIC column [4],
which separates glycans by hydrogen bonding, resulting in a
size and composition-based separation. This type of column
is particularly useful for the separation of glycans released
from monoclonal antibodies, of which the majority are
neutral [5].
Based on novel mixed-mode surface chemistry, the
GlycanPac AXH-1 column combines both weak anion-
exchange (WAX) and HILIC retention mechanisms for
optimal selectivity and high resolving power [6]. The WAX
functionality provides retention and selectivity for negatively
charged glycans, while the HILIC mode facilitates the
separation of glycans of the same charge according to their
polarity and size. As a result, the GlycanPac AXH-1 column
