4
Results and Discussion
The protocol outlined in this application note yields detailed
information on the set of glycans present in proteins
including mAbs. The protocol describes a fully integrated
workflow that combines novel column technology
(GlycanPacAXH-1 column), mass spectrometry (Q Exactive
mass spectrometer), and a bioinformatics tool (SimGlycan
software). This fully integrated workflow is demonstrated
for
N
-glycans released from bovine fetuin glycoprotein, but
can be used for released
N
-glycans from any glycoprotein.
The GlycanPac AXH-1 column described in this application
note can be used for qualitative and quantitative
characterization of neutral and charged glycans present on
proteins. The elution of glycans is based on charge: the
neutral glycans elute first, followed by the separation of
acidic glycans from mono-sialylated to penta-sialylated
species. Glycans of each charge state are further separated
based on their size and polarity. Separation of glycans based
on charge, size, and polarity–combined with MS–provides
complete structural and quantitative information.
2AB labeled
N
-linked glycans from bovine fetuin were
separated on the GlycanPac AXH-1 column and analyzed on
a Q Exactive mass spectrometer (Figure 1). Data-dependant
MS/MS spectra were acquired on all precursor ions (z
≥
2),
and SimGlycan software was used for structural elucidation.
A representative example of the analysis is shown in
Figure 2. The Q Exactive mass spectrometer was selected
for these experiments because of its 140,000 FWHM
resolution at
m/z
200, high scan speeds at all resolution
settings, and sensitivity. All of these contribute to the
detection of minor glycan species and generation of high-
quality MS/MS spectra even for low-abundance glycans.
Additionally, the Q Exactive mass spectrometer has the
ability to generate higher-energy collisional dissociation
(HCD) with high-resolution, accurate-mass (HR/AM)
fragment ions. This allows for differentiation of near-mass
fragment ions, which were observed to be useful for correctly
assigning branching and linkage. The variation of collision
energy can provide different fragment ions within the mass
spectrometer. To maximize both glycosidic and cross-ring
fragments, normalized stepped collision energy (NSCE) was
incorporated. This provided optimum conditions for
generation of a maximum number of both cross-ring and
glysodic cleavages in a single spectrum, thereby increasing
confidence in the identification (Figure 2). The detailed
structural information obtained from the MS/MS data
shown in Table 2 further validated the ability of the
GlycanPac AXH-1 column to separate glycans based on
charge, size, and polarity.
The use of LC-MS/MS for glycan analysis increases the
complexity of data analysis due to the large number of
MS/MS spectra generated. SimGlycan software was
incorporated to simplify data analysis.
6,7
SimGlycan software
predicts the structure of a glycan from the MS
n
data.
It accepts the raw MS
n
files, matches them with its own
database of theoretical fragmentation of over
22,000 glycans, and generates a list of potential glycan
structures. Each proposed structure is assigned a score to
reflect how closely it matches with the experimental data.
Source Conditions
Source position
C
Sheath gas flow rate (arb units)
20
Auxillary gas flow rate (arb units)
5
Sweep gas flow rate
0
Spray voltage (kV)
3.30
Capillary temperature (ºC)
275
S-lens RF level
50
Heater temperature (ºC)
300
Data Processing and Software
Chromatographic
Thermo Scientific
™
Chromquest
™
software
v 5.0 Chromatography Data
System
MS data acquisition
Thermo Scientific
™
Xcalibur
™
software v 2.2 SP1.48
MS/MS data analysis
SimGlycan software v 4.5
SimGlycan Search Parmeters
Ion mode
Negative
Adduct
H
Chemical derivatization
Underivatized
Match fragment ion
< Precursor
m/z
charge state
for charge state
Precursor ion
m/z
10 ppm
Fragment ion
0.05 Da
Modification
2AB
Class
Glycoprotein
Sub class
N
-glycan (Intact Core)
Biological source
Bovine Fetuin
Pathway
Unknown
Search structure
All
Glycan type
All
% of evident glycosidic
2
linkages
Fragmentation pattern
Specify Expected Fragments
in the Spectra
Glycosidic
B: Yes; C: Yes; Y:Yes; Z:Yes
Cross-ring
A:Yes; X:Yes
Glycosidic/Glycosidic
Z/Z: Yes; Y/Y: Yes;
B/Y or Y/B: Yes; C/Z or Z/C: No;
Z/Y or Y/Z: No; B/Z or Z/B: No;
C/Y or Y/C: Yes
Cross-ring/Glycosidic
A/Y or Y/A: Yes; A/Z or Z/A:Yes;
X/Y or Y/X: Yes; X/Z or Z/X: No;
X/B or B/X: Yes; X/C or C/X: Yes
