Concanavalin A Column for Analysis
of Glycoproteins and Their Tryptic
Glycopeptides
Dai Zhenyu,
1
Xu Qun,
1
and Jeffrey Rohrer
2
1
Thermo Fisher Scientific, Shanghai, People’s Republic of China;
2
Thermo Fisher Scientific, Sunnyvale, CA, USA
Application Note 1014
Key Words
Off-Line 2D HPLC, Con A Enrichment, Reversed-Phase Peptide Mapping,
UltiMate 3000 ×2 Dual Biocompatible Analytical LC System, Lectin Affinity
Chromatography
Introduction
Glycosylation, one of the most common and complex
post-translational modifications, is of great interest for
its role in many biological processes such as molecular
recognition, cell signaling, and immune defense. Addition-
ally, glycosylation has profound effects on the properties
of a glycoprotein including solubility, immunogenicity,
circulatory half-life, and thermostability. These protein
properties are important in today’s biotechnology industry
because proteins are produced for human therapeutics.
For in-depth research of a glycoprotein, peptide mapping
is necessary. Several factors make glycopeptide research by
mass spectrometry (MS) challenging. First, glycopeptides
usually constitute a minor part of the glycoprotein’s total
peptides. Second, the MS signal intensities of the glyco-
peptides are lower relative to nonglycosylated peptides
due to lower ionization efficiency and heterogeneous
glycan structures on the same glycosylation site. Third, a
glycopeptide’s MS signal can be suppressed by nonglyco-
sylated peptides. Therefore, enrichment of glycopeptides
from a complex peptide mixture is desired and lectin
affinity capture technology has been developed to fulfill
this purpose.
Among all lectins, Concanavalin A (Con A) is the most
well characterized and widely used. It can capture
glycoproteins containing asparagine-linked (
N
-linked)
high-mannose type glycans,
N
-linked hybrid type glycans,
and some
N
-linked biantennary complex type glycans.
The Thermo Scientific ProSwift ConA-1S Affinity Column
was designed to isolate glycans, glycopeptides, or
glycoproteins from complex samples. But unlike other
Con A columns, it is built upon a monolithic support
and is designed to be used on a high-performance liquid
chromatography (HPLC) system. HPLC compatibility and
the monolithic support deliver multiple advantages: faster
separation, better sample recovery, high peak efficiency,
and high-throughput capability.
This approach also enables an automated configuration
to identify glycopeptides using the ProSwift
™
ConA-1S
Affinity column to extract the glycopeptides from a
peptide mixture. The isolated glycopeptides can then be
separated by reversed-phase HPLC, and their identity
confirmed by selected ion monitoring (SIM) of diagnostic
sugar oxonium ions (e.g.,
m/z
204, 366, and 163).
This method requires only a single quadrupole mass
spectrometer.
Goal
The goals of this work are to:
• Use the ProSwift ConA-1S Affinity column to purify
glycoproteins from commercial preparations of
those glycoproteins.
• Extract glycopeptides from the tryptic digests of
purified horseradish peroxidase (HRP), ovalbumin,
and ribonuclease B glycoproteins.
• Identify individual glycopeptides of a tryptic digest
after glycopeptide extraction using reversed-phase
separation.
• Use a Thermo Scientific Dionex UltiMate 3000 ×2
Dual Biocompatible Analytical LC system to
automate the experiment.
