N-linked Glycan Analysis using cHiPLC® System - Eksigent

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
p 2