Application Note 1014
AN70125_E 08/12S
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Endo H Digestion Eliminates the Con A Binding
Ability of HRP
Endo H has been reported to cleave within the chitobiose
core of high-mannose type and some hybrid type oligosac-
charides from
N
-linked glycoproteins. Figure 6 shows
that HRP cannot be retained by the ProSwift ConA-1S
Affinity column after Endo H treatment. This observation
confirms that main glycosylation types of HRP are high
mannose and/or hybrid. Peak 3 in Figure 6 is postulated
to be either the released oligosaccharides—because it is
not found in the Endo H control (no HRP added)—or
more likely, the fraction of glycosylated HRP that is
not susceptible to Endo H (i.e., all Endo H-susceptible
structures have been removed and only the nonsusceptible
structures remain on the HRP). It is more likely a
non-Endo H-susceptible fraction because oligosaccharides
have little or no absorbance at 210 nm.
Conclusion
This work shows that the HPLC-compatible ProSwift
ConA-1S Affinity column can capture glycoproteins and
glycopeptides efficiently. The UltiMate 3000 ×2 Dual
Biocompatible Analytical LC system can automate the
entire off-line 2D process from ProSwift ConA-1S Affinity
column sample enrichment and fraction collection to
automatic reanalysis of collected sample by peptide
mapping. Monitoring oxonium ions (e.g.,
m/z
204, 366,
and 163) in a peptide mixture with a single quadrupole
mass spectrometer is a selective, sensitive, and reliable
method that also confirms the identity of glycopeptides
captured by the ProSwift ConA-1S Affinity column.
References
1.Dionex (now part of Thermo Scientific) Application
Update 183: Separation of Peptides from Enzymatic
Digestion on Different Acclaim Columns: A
Comparative Study. Sunnyvale, CA, 2011. [Online]
-
Peptides-EnzyDigest-AcclaimCompar-02Nov2011-
LPN2973.pdf (accessed June 12, 2012).
2.Harvey, D.J.; Wing, D.R.; Küster, B.; Wilson, R.B.H.
Composition of N-Linked Carbohydrates from
Ovalbumin and Co-purified Glycoproteins.
J. Am. Soc.
Mass Spectrom.
2000
,
11
, 564–571.
3.Wuhrer, M.; Hokke, C.H.; Deelder, A.M. Glycopeptide
Analysis by Matrix-Assisted Laser Desorption/
Ionization Tandem Time-of-Flight Mass Spectrometry
Reveals Novel Features of Horseradish Peroxidase
Glycosylation: A Correlation Study.
Rapid Commun.
Mass Spectrom.
2004
,
18
, 1741–1748.
4. Bean, M.F.; Annan, R.S.; Hemling, M.E.; Mentzer, M.;
Huddleston, M.J.; Carr, S.A. LC-MS Methods for
Selective Detection of Posttranslational Modifications in
Proteins: Glycosylation, Phosphorylation, Sulfation,
and Acylation. In
Techniques in Protein Chemistry
;
J. Crabb, ed.; Academic Press: San Diego, 1995; pp
107–116.
5.Wuhrer, M.; Balog, C.R.A.; Koeleman, C.A.M; Deelder,
A.M; Hokke, C.H. New Features of Site-Specific
Horseradish Peroxidase (HRP) Glycosylation
Uncovered by nano-LC-MS with Repeated Ion-
Isolation/Fragmentation Cycles.
Biochim. Biophys. Acta
2005
,
1723
, (1-3) 229–239.
Figure 6. Glycosylated HRP enrichment on the ProSwift ConA-1S Affinity column
(A) before and (B) after Endo H treatment.
A
1
2
B
3
2
-50
400
0
5
10
15
20
25
mAU
Minutes
300
-50
mAU
Column:
ProSwift ConA-1S Affinity (5 × 50 mm)
Mobile Phase:
A: 50 mM sodium acetate, 200 mM sodium chloride, 1 mM calcium
chloride, pH 5.3
B: 100 mM
α
-methyl mannoside in mobile phase A
Gradient:
0–5.0 min, 0% B; 5.0–5.5 min,
0–100% B; 5.5–15 min, 100% B
Flow Rate:
0.5 mL/min
Inj. Volume:
20 µL
Temperature:
30 °C
Detection:
UV at 214 nm
Sample Preparation: 1 mg/mL HRP in water/mobile phase A
Samples:
A
. Before Endo H treatment
B
. After Endo H treatment
Peaks:
1. HRP
2., 3. Deglycosylated HRP
