N-linked Glycan Analysis using cHiPLC® System - Eksigent

N-linked Glycan Analysis using cHiPLC ® System
Xiang Zhu 1 , Justin Hyche 2 , Remco van Soest 1
1 Eksigent, part of AB SCIEX, Dublin, CA; 2 ProZyme, Hayward, CA
Glycosylation is known as one of the most important protein
post-translational modifications in eukaryotic cells. Glycans are
involved in a number of biological processes such as cell
adhesion, immune recognition, and protein regulation. Abnormal
glycosylation has been observed in many types of cancers. One
of the most commonly emergent changes in glycosylation
observed in cancer cells is the increasing branching on complex
N-linked glycans. Therefore, understanding the changes in
glycan profiles on proteins has become very important for
biomarker studies. LC-MS has been recognized as a useful
technique to qualitatively and quantitatively analyze glycan
structures.
The separation and analysis of glycoprotein released glycans
using reversed phase LC-MS is always a challenging task
because these molecules are usually very hydrophilic, difficult to
ionize and contain a variety of complex compositions, branching
and linkage isomers. Herein, a chip based nanoLC system, the
cHiPLC ® system 1 , was used to separate and identify N-linked
glycans from some standard glycoproteins. The glycan
separation utility of both hydrophilic interaction chromatography
(HILIC) and porous graphitic carbon (PGC) chip columns was
investigated.
Figure 1. Extracted Ion Chromatogram of N-Glycans Released from
Bovine Fetuin using a HILIC cHiPLC ® Column. N-linked glycans from
bovine fetuin are composed of mono-, di-, tri- and tetra-sialylated glycans,
which are challenging to separate and analyze. The HILIC cHiPLC ®
column can completely resolve the biantennary and triantennary N-
glycans, including positional isomers that differ only in the sialic acid
linkage positions.
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Key Features of the cHiPLC ® System
Plug & Play simplicity
Expert results for non-experts
Cost and time savings
Switch workflows easily in multi-user labs
Workflow flexibility
Direct injection
Trap and Elute – dirty samples
Dual column multiplexing – increasing throughput 2
Serial 2-column – increasing peak capacity and depth
of proteome coverage 3
Variety of phases available (C18, C4, HILIC, PGC, etc.)
Any lab, any person, any MS
A new level of reproducibility
Improved robustness for increased uptime
Multi-lab projects now a reality

Experimental
Sample Preparation: Underivatized N-glycan mixtures from
bovine fetuin and human immunoglobulin G (IgG) (ProZyme,
Hayward, CA) were used to test the two separation phases.
Samples were freshly dissolved in 20% mobile phase A/80%
mobile phase B solution for HILIC analysis and 100% mobile
phase A for PGC separation.
LC-MS/MS: An Eksigent NanoLC-Ultra ® 1D plus system
(Eksigent, part of AB SCIEX Dublin, CA, USA) was used in
combination with a cHiPLC ® system (Eksigent) in direct inject
mode. The NanoLC-Ultra ® system delivers the solvent based on
binary gradient pumps that use patented Microfluidic Flow
Control (MFC) pump technology. The porous graphitic carbon
(PGC) cHiPLC ® column was packed with 3 µm graphitized
carbon. The hydrophilic interaction chromatography (HILIC)
cHiPLC ® column contained 2.7 µm HALO Fused-Core particles
as packing material (Advanced Materials Technology,
Wilmington, DE, USA). The MS data was acquired using an LTQ
ion trap mass spectrometer (Thermo Fisher, San Jose, CA,
USA). Detailed experimental conditions were listed in Table 1.
Glycan Separation Comparisons
Glycans are typically hydrophilic and thus not retained well by
reversed phase liquid chromatography. Common alternative
column phases include porous graphitic carbon (PGC) and
hydrophilic interaction chromatography (HILIC). Both of these
column phases retain hydrophilic compounds more than typical
reversed phase chromatography and result in longer retention
times vs. reversed phase LC. Therefore PGC and HILIC columns
are often applied for resolving complex glycan mixtures.
Figure 2. Extracted Ion Chromatogram of N-Glycans Released from
Bovine Fetuin using cHiPLC ® PGC Column. Compared to C18 phase,
the PGC column retains the hydrophilic molecules much better and
separates the positional isomers as well. The tetra-sialylated glycans are
not separated as well as with the HILIC column (Figure 1), possibly due
to their strong interaction with the PGC stationary phase.
Figure 1 shows an example of using a HILIC-nanoLC/MS
approach for the separation and detection of N-linked glycans
from bovine fetuin. This particular sample contains a mixture of
mono-, di-, tri-, and tetra-sialylated glycans, making the
separation very challenging. Separation and identification of
these N-glycans was also performed using a PGC cHiPLC ®
column (Figure 2). One of the advantages of using PGC
compared to HILIC is that the sample can be dissolved in high
aqueous solutions to reduce sample loss. Bi-antennary and triantennary
structures are well separated including the isomeric
species. Compared to the HILIC cHiPLC column, the tetrasialylated
glycans give broader peaks using the PGC column,
Table 1. LC-MS Conditions for N-Glycan Analysis.
HILIC Fetuin N-glycan PGC Fetuin N-glycan HILIC IgG N-glycan PGC IgG N-glycan
Sample Concentration 2 ng/µL 2 ng/µL 20 ng/µL 2 ng/µL
Injection Volume 1 µL 1 µL 1 µL 1 µL
Column
Halo HILIC cHiPLC ®
75 µm x 15 cm
PGC cHiPLC ®
75 µm x 15 cm
Halo HILIC cHiPLC ®
75 µm x 15 cm
PGC cHiPLC ®
75 µm x 15 cm
Mobile Phase A
50mM ammonium formate
pH 4.0
0.08% ammonia
pH 10.0
50mM ammonium formate
pH 4.0
Water (0.1% formic acid)
pH 4.0
Mobile Phase B
Acetonitrile
(0.1% formic acid)
Acetonitrile
(0.1% formic acid)
Acetonitrile
(0.1% formic acid)
Acetonitrile
(0.1% formic acid)
Temperature 60 °C 60 °C 60 °C 60 °C
Detection LTQ, negative mode LTQ, negative mode LTQ, positive mode LTQ, positive mode
Flowrate 300 nL/min 300 nL/min 300 nL/min 300 nL/min
Gradient 70-60% B in 60 min 5-50% B in 30 min 90-60% B in 40 min 3-40% B in 40 min
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The detection using the HILIC cHiPLC ® column also identified
most of the major IgG N-glycans, as shown in Figure 5. Some
glycans were only detected either in HILIC or PGC conditions,
making the two methods complementary to each other.
Figure 3. Identified N-linked Glycans Structures from IgG. Most of
these glycans were identified using both cHiPLC ® columns. Some were
detected either in HILIC or PGC LC-MS only as indicated by the numbers
labeling the peaks in Figures 4 and 5.
possibly due to stronger interaction with the PGC stationary
phases.
As shown in Figure 4, a mixture of high mannose, complex and
hybrid N-linked glycans released from human IgG were well
separated using PGC cHiPLC ® column. Different isomers were
resolved as well and shown as two separated peaks.
Figure 5. Extracted Ion Chromatogram of Human IgG N-Glycans
using HILIC cHiPLC ® Column. Some glycans were only identified in
this method, making it complementary to PGC analysis.
Conclusions
Two nanoflow cHiPLC ® columns (HILIC and PGC) were coupled
with a high performance NanoLC-Ultra ® system and utilized for
the analysis of hydrophilic carbohydrates. Both methods
separated complex mixtures of N-glycans including some
isomeric species, enabling direct identification of the glycans by
LC/MS.
References
1. cHiPLC ® -nanoflex System: Making nanoLC/MS Easier and
More Reproducible. Eksigent Product Note 0910-1000
Figure 4. Extracted Ion Chromatogram of Human IgG N-Glycans
using PGC cHiPLC ® Column. The peak number and corresponding
structures were listed in Figure 3. A combination of high mannose,
complex and hybrid N-glycans were separated using graphitized carbon
chip column at nanoflow conditions. Different isomers were also
resolved and split into two peaks.
2. Increasing Throughput of nanoLC using Two-Column
Switching Workflows. AB SCIEX Technical Note 1870411-
01
3. Increasing Depth of Coverage using Serial Two-Column
Workflows. AB SCIEX Technical Note 1870211-02
For Research Use Only. Not for use in diagnostic procedures.
© 2012 Eksigent, a division of AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX TM is being used under license. All
rights reserved. Information subject to change without notice.
Patents pending worldwide. Printed in the USA. 6540212-01
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