3
Separation Conditions
Column:
GlycanPac AXH-1, 2.1 x 150 mm, 1.9 µm
082472
Mobile phase A:
Acetonitrile / water (80:20, v/v)
Mobile phase B:
Ammonium formate (80 mM, pH 4.4)
Column temperature:
30 °C
Sample volume:
1 µL
Gradient:
Time
%A
%B Flow Rate Curve
(min)
(mL/min)
-10
97.5
2.5
0.4
5
0
97.5
2.5
0.4
5
30
87.5
12.5
0.4
5
35
75.0
25.0
0.4
5
40
62.5
37.5
0.4
5
MS Conditions
MS instrument:
Q Exactive hybrid quadrupole-Orbitrap MS
Ionization mode:
Negative ion mode
MS scan range:
380-2000
m/z
Resolution:
70,000
AGC target
1 x 10
6
Max IT:
60 ms
dd-MS2 resolution:
17,500
MS/MS AGC target
2 x 10
5
MS/MS max IT:
1000 ms
Isolation window:
2
m/z
Dynamic exclusion:
90 s
Data Processing and Software
Chromatographic software:
Thermo Scientific™ ChromQuest™ Chromatography Data System
version 5.0
MS data acquisition:
Thermo Scientific™ Xcalibur™ software version 2.2 SP1.48
MS/MS data analysis:
SimGlycan
®
software (PREMIER Biosoft)
Results
Glycan Separation by Charge, Size and Polarity
Figure 1 shows the separation of neutral and acidic 2AB labeled
N
-glycans from bovine fetuin
using a GlycanPac AXH-1 (1.9 μm, 2.1 × 150 mm) column. The glycan elution profile consists of a
series of peaks grouped into several clusters in which the neutral glycans elute first, followed by
monosialylated, disialylated, trisialylated, tetrasialylated, and finally pentasialylated species.
Analytes in each cluster represent glycans of the same charge. Within each cluster, the glycans
having the same charge are further separated according to their sizes and polarity by HILIC
interaction. The structure of the glycans present in each peak was determined in an LC-MS/MS
study as shown in the following section.
Structural elucidation
The 2AB labeled
N
-glycans from bovine fetuin were separated on the GlycanPac AXH-1 column
based on the separation conditions using a two eluent system and analyzed on a Q Exactive
benchtop mass spectrometer. The total ion chromatogram (TIC) is shown in Figure 1. For
structural elucidation, data dependant MS/MS spectra were acquired on all precursor ions (z ≤ 2)
and SimGlycan software from PREMIER Biosoft was used for data analysis [8]. The detailed
structural information obtained (Table 1) from the MS/MS data further validated the ability of
GlycanPac AXH-1 columns to separate glycans based on charge, size, isomers, and polarity. These
results also confirmed that the GlycanPac AXH-1 column would be ideal for MS use.
