provides unparalleled capabilities for glycan separations. In addition, this column has the flexibility
to be used in a purely ion-exchange, charge-based separation mode for the separation of various
glycans without discrimination of size and polarity. This makes it a suitable tool for accurate
quantification of glycans based on charge, which cannot be achieved with any other HPLC/UHPLC
columns on the market. The GlycanPac AXH-1 column is designed for use with LC-fluorescence
detection and LC/MS applications using volatile aqueous buffers (e.g., ammonium acetate or
ammonium formate) and acetonitrile. The substrate of the GlycanPac AXH-1 column is based on
high purity 1.9 μm and 3.0 μm spherical silica for UHPLC and standard HPLC applications,
respectively.
In this application note we highlight the use of a GlycanPac AXH-1 column and Q Exactive hybrid
quadrupole-Orbitrap mass spectrometer for the structural analysis of a 2-aminobenzamide (2AB)
labeled
N
-glycans from bovine fetuin.
2
Experimental Details
Consumables
Part Number
GlycanPac AXH-1, 1.9 µm, analytical column (2.1 × 150 mm)
082472
Deionized (D.I.) water, 18.2 MΩ-cm resistivity
Fisher Scientific™ HPLC grade acetonitrile (CH
3
CN)
AC610010040
Fisher Scientific LC/MS grade formic acid
A117-50
Fisher Scientific ammonium formate
AC40115-2500
Thermo Scientific Premium 2 mL vial convenience kit
60180-600
New England Biolabs
®
PNGase F
P0705L
Bovine fetuin from a commercial source
Thermo Scientific™ Hypercarb™ 6 mL cartridge
60106-403
Fisher Scientific trifluoracetic acid
28904
Fisher Scientific sodium cyanoborohydride
AC16855-0500
Fisher Scientific anthranilamide (2AB)
AC10490-5000
Fisher Scientific glacial acetic acid
AA36289AP
Equipment
Part Number
Thermo Scientific™ Dionex™ UltiMate™ 3000 Bio-RS system, consisting of LPG-3400RS pump,
6078.4330
TCC-3000RS thermal compartment, WPS-3000TRS pulled-loop well plate autosampler, FLD3400RS
fluorescence detector with Dual-PMT, and a 2 µL micro flow cell
Q Exactive hybrid quadrupole-Orbitrap mass spectrometer
Thermo Scientific™ SpeedVac™ system
Thermo Scientific Lyophilizer (Labconco
®
FreeZone
®
-105 °C 4.5 L benchtop freeze dry system)
16-080-207
Thermo Scientific 24-port SPE vacuum manifold
60104-233
Buffer Preparation
Ammonium formate (80 mM, pH 4.4): Dissolve 5.08 ± 0.05 g of ammonium formate (crystal) and 0.60 g of formic acid in
999.6 g of D.I. water. Sonicate the resulting solution for 5 minutes.
Sample Preparation
1. Native
N
-glycans are released from glycoproteins with PNGase F enzyme and purified by a Hypercarb cartridge
(6 mL) with the help of 24-port SPE vacuum manifolds under vacuum. The released glycans are conjugated with a
2-amino benzamide (2AB) label group using the reported procedure of Bigge et al. [7].
2. Dissolve 2AB labeled
N
-glycan from fetuin (5,000 pmol) in 25 µL D.I. water in a 250 µL autosampler vial.
3. Add 75 µL acetonitrile to the same vial and mix till uniformity.
Note: Store the standard at -20 °C.
