High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry

Full text of DOI:10.1002/anie.200461685

Angewandte
Chemie
[
1]
and within the cells. This highly dynamic process in which
rapid changes in the carbohydrate structures occur in
response to cellular signals or cellular stages result in the
key informational markers of some serious human diseases.
For example, it is known that carbohydrates of IgG can be
drastically altered to form unusual structures in patients with
[
2]
rheumatoid arthritis and specific carbohydrates are used as
[
3]
tumor-associated markers in pancreatic and colon cancers.
Recently, it has also been reported that the oligosaccharides
of the therapeutic human IgG play a critical role of enhancing
[
4]
antibody-dependent cellular cytotoxicity. Glycosylation of
nuclear and cytoplasmic proteins with a GlcNAc residue is
ubiquitous in nearly all eukaryotes and is a crucial event in
posttranslational modifications for regulating protein func-
[
1b]
tions within the cells. Dynamic perturbations in O-GlcNAc
regulation have been implicated in the etiology of type II
[
5]
diabetes, cancer, and neurodegenerative diseases.
Analytical Methods
Although there have been substantial advances in our
understanding of the effects of glycosylation on some
biological systems, we still do not fully understand the specific
functional roles of carbohydrates and the relationship
between their structures and functions. The major difficulty
in carbohydrate sequencing is a consequence of the fact that
the purification of trace amounts of oligosaccharides requires
extremely tedious multistep processes. This is because crude
sample mixtures prepared by enzymatic digestion from cells,
organs, serum, etc. usually contain large amounts of impur-
ities such as peptides, lipids, and salts. These technical
problems in the sequencing of carbohydrates make it
impossible to achieve high-throughput protein glycomics.
We report herein that the combined use of two novel
techniques, chemoselective glycoblotting and MALDI-TOF/
High-Throughput Protein Glycomics: Combined
Use of Chemoselective Glycoblotting and
MALDI-TOF/TOF Mass Spectrometry**
Shin-Ichiro Nishimura,* Kenichi Niikura,
Masaki Kurogochi, Takahiko Matsushita,
Masataka Fumoto, Hiroshi Hinou, Ryousuke Kamitani,
Hiroaki Nakagawa, Kisaburo Deguchi, Nobuaki Miura,
Kenji Monde, and Hirosato Kondo
Glycosylation is one of the posttranslational modifications of
proteins in eukaryotes. This step is thought to modulate a
wide range of protein functions both on the cellular surfaces
[
6]
TOF mass spectrometry, allows for both facile purification
and precise analysis of common oligosaccharides and glyco-
peptides from native glycoproteins.
[
*] Prof. S.-I. Nishimura, Dr. K. Niikura, Dr. M. Kurogochi,
T. Matsushita, R. Kamitani, Dr. H. Nakagawa, Dr. K. Deguchi,
Dr. N. Miura, Dr. K. Monde
The concept of the “sugar family tree,” constructed by
[
7]
Fischer, motivated us to use the chemoselective “glycoblot-
ting” technique, which has become a key feature in the
efficient isolation of carbohydrates in this study. Fischer found
that the reaction of glucose, mannose, and major oligosac-
charides with phenylhydrazine proceeds smoothly to give the
corresponding stable phenylhydrazone derivatives under mild
basic and aqueous conditions. Once oligosaccharides are
released from glycoconjugates, they can be regarded as a
general class of the compound library which have an aldehyde
or ketone group at each reducing terminal. As indicated in
Scheme 1a, aldehydes preferentially react with reagents
bearing hydrazine-like functional groups. Fischer-type
reagents do not need any catalyst or reducing agent to
accelerate the reaction with sugars and the reactions usually
proceed under mild conditions. However, the reactions of
aldehydes with primary amino groups require some activating
reagent for the formation of stable products. As a result,
carbohydrates preferentially react with Fischer-type reagents
even in the presence of large amounts of peptides or amino
acids with primary amino groups.
Division of Biological Sciences
GraduateSchool of Sci en ce
Frontier Research Center for Post-Genome Science and Technology
Hokkaido University
N21, W11, Sapporo 001-0021 (Japan)
Fax: (+81)11-706-9042
E-mail: shin@glyco.sci.hokudai.ac.jp
Prof. S.-I. Nishimura, M. Fumoto, Dr. H. Hinou
Research Center for Glycoscience
National Institute of Advanced Industrial Science and
Technology (AIST)
Sapporo 062-8517 (Japan)
Dr. H. Kondo
Discovery Research Laboratories
Shionogi & Co. Ltd.
Osaka 553-0002 (Japan)
[
**] This work was partly supported by a grant for the National Project
on “Functional Glycoconjugate Research Aimed at Developing for
New Industry” from the Ministry of Education, Science, and Culture
of Japan. Part of this study was presented at the Annual Conference
of the Society for Glycobiology, December 3–6, 2003, San Diego, CA
(
USA).
The unique chemical characteristics of the sugar family
encouraged us to design novel polymers for capturing only
carbohydrates from crude samples on the basis of the
Supporting information for this articleis availableon theWWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2005, 44, 91 –96
DOI: 10.1002/anie.200461685
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
91

Communications
Scheme 1. Fischer’s concept and glycoblotting by Fischer-type polymer reagents. a) Reactions of carbohydrates with Fischer’s reagents. b) Synthe-
ses and reactions of Fischer-type polymer reagents: 1) liposomes prepared with phosphatidylcholine-related monomer, then UV (254 nm) irradia-
tion, 2) radical copolymerization (ammonium persulfate) with acrylamide, followed by deprotection with 4n HCl.
chemoselective ligation strategy. From considerations of the
chemical stability, the handling of reagents, and the feasibility
to the general synthesis of versatile polymer materials, we
have developed several Fischer-type polymer reagents with
reactive and stable oxylamino functional groups by polymer-
ization of a diacetylene-containing lipid derivative 1 or an
acrylamide-type derivative 2 (Scheme 1b). Liposomes com-
posed of diacetylene-containing reagent 1 and phospholipid
derivatives can be readily polymerized by UV irradiation to
produce polymer-based nanoparticles having a mean diame-
ter of 200–300 nm. Compound 2 gives a water-soluble
polymer on radical copolymerization with acrylamide. We
evaluated the feasibility of the polymers displaying oxylamino
functional groups as a useful platform for the “trap and
release” of the target oligosaccharides and glycopeptides.
The general protocol for the isolation and analysis of
carbohydrates proposed here is simple and easy: a) Trapping
carbohydrates by mixing polymers with unpurified proteo-
lytic digest; b) collecting polymers by spin filtration from a
crude mixture of peptides, salts, and other impurities; and
c) releasing and subsequent MALDI-TOF MS analysis of the
target carbohydrates (Figure 1). In this study we selected the
N-glycans of human IgG to test the efficiency of protein
glycomics in the strategy, since the importance of substantial
structural alteration of IgG N-glycans has been extensively
[
3,8]
investigated, The structural characterization of human IgG
N-glycans was carried out using crude IgG samples obtained
from human serum (100 mL, Figure 1). The merits of glyco-
blotting are evident since MALDI-TOF MS analysis of
oligosaccharides trapped on the polymer has proven to be
remarkably improved by using trapping and spin filtration
(Figure 2b). When the crude digest was directly subjected to
MALDI-TOF MS analysis we obtained an extremely com-
plex spectrum (Figure 2a). The precursor ion peaks, which
correspond to the N-glycans, cannot be distinguished from
numerous other signals in the spectrum because of an excess
amount of peptide impurities. As anticipated, highly sensitive
and singly charged precursor ion peaks were generated in the
presence of a-cyano-4-hydroxycinnamic acid (CHCA) and
these greatly facilitated the subsequent sequencing and
quantitative analysis of the oligosaccharides. In addition, it
was found that the fragmentation of the precursor ions by the
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Figure 1. General procedure for chemoselective glycoblotting
and MALDI-TOF mass analysis of carbohydrates.
[
6]
LIFT-TOF/TOF method allowed for the efficient and
precise sequencing of N-glycans. The TOF/TOF spectral
patterns of the product ion peaks could then be applied for
further data fitting with the known mass database of known
oligosaccharides. Fragmentation by the TOF/TOF technique
of an ion peak at m/z 1486 provided the typical fragment ion
series corresponding to that of a known pyridyl-aminated
[
9]
oligosaccharide (Figure 2c).
This result indicated that
carbohydrate mass fingerprinting (CMF) analysis by the
MALDI-TOF/TOF method allows for the precise structural
identification of oligosaccharides captured from glycopro-
teins by glycoblotting.
The CMF analysis based on TOF/TOF can also be used to
determine the ratio of oligosaccharide isomers, as shown in
the analysis of the ion peak at m/z 1648 (see Figure 3). The
observed fragmentation of this precursor ion peak was found
to be a mixture of two structural isomers and the analysis
using the correlation coefficient calculated by using the
known compounds enabled the ratio of the two isomers to be
estimated as 62:38.
It was clearly suggested that the structure of each
oligosaccharide measured by the MALDI-TOF instrument
was in good agreement with the data obtained by a conven-
tional HPLC-based analysis of the corresponding pyridyl-
[
10]
aminated derivatives prepared from human IgG.
The
relative intensity of each carbohydrate response given by
the two methods exhibited quite similar results (see the
Supporting Information), which suggests that the polymer
captures carbohydrates with a reaction efficiency in propor-
tion to the amount of each oligosaccharide existing in the
sample mixtures. This result means that glycoblotting is a
general and versatile method for trapping a variety of
carbohydrates bound to proteins.
Our attention was next directed toward the application of
the glycoblotting strategy for the identification of the O-
GlcNAc site present in abundance in nucleocytoplasmic
[
1b]
glycoproteins. Recently, we succeeded in the high-through-
put identification of O-glycosylation (O-GalNAc) sites by
high-speed analysis in the LIFT-TOF/TOF mode of the ion
peaks of the glycosylated products in the presence of 2,5-
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[
6a]
dihydroxybenzoic acid (DHB).
To test the feasibility of
glycoblotting and the subsequent TOF/TOF analysis of
glycopeptides, we preliminarily investigated the enzymatic
modification of
a synthetic glycopeptide (PSVPVS(O-
GlcNAc)GSAPGR) which was identified as the O-GlcNAc-
[
11]
modified peptide derived from basic phosphoprotein. This
glycopeptide was subjected to: a) Galactosylation with gal-
[
12]
actosyl transferase (GalT) and b) oxidation with galactose
oxidase (GOT) to generate a terminal aldehyde group at the
C-6 position of the modified galactose residue (Scheme 2). A
Scheme 2. Strategy for the analysis of O-GlcNAc modified peptides.
crude glycopeptide mixture was directly used for further
blotting with polymer reagent 2 and the subsequent analysis
by MALDI-TOF mass spectrometry. Since peptide fragments
bearing oligosaccharides prepared by tryptic digestion often
exhibited poor solubility in water, water-soluble Fischer-type
reagents may be a useful alternative to nanoparticles in terms
of the efficiency. The TOF/TOF MS spectrum of the
precursor ion at m/z 1473.71 measured with DHB gave
highly sensitive and ideal fragmentation patterns because of
the glycopeptide containing a (CHO)Galb!4GlcNAcb!Ser
residue (Figure 4b). As anticipated, fragmentation in the
presence of the DHB matrix occurred preferentially at
peptide bonds without serious de-O-glycosylation (elimina-
tion) or fragmentation (cleavage) in the carbohydrate moiety.
This characteristic greatly facilitated the identification of the
O-glycosylation site in the target molecule (Figure 4c). This
result suggests that the aldehyde attachment by enzymatic
derivatization of the O-GlcNAc-modified peptide enables a
high-throughput identification of O-glycosylation sites based
Figure 2. MALDI-TOF analysis of human IgG N-glycans. a) MALDI-
TOF mass spectrum of crude N-glycans before glycoblotting. b) Major
precursor ion peaks of N-glycans obtained by glycoblotting. c) Frag-
mentation by MALDI-LIFT-TOF/TOF tandem mass spectroscopy. Frag-
mentation of the precursor ion at m/z 1486 was performed according
to the conditions reported previously in the presence of CHCA as a
[
13]
on the glycoblotting method.
The merit of the present
strategy is evident because the identification of new O-
GlcNAc-modified proteins and the sites of modification
would facilitate more global studies of the regulatory role of
this posttranslational modification.
[
11]
matrix. Precise structural identification was carried out by comparing
the fragmentation pattern of the known pyridyl-aminated oligosacchar-
ide. All measurements were performed using an Ultraflex TOF/TOF
mass spectrometer equipped with a reflector, and controlled by the
Flexcontrol 1.2 software package (Bruker Daltonics GmbsH, Bremen,
Germany). Ions generated by a pulsed UV laser beam (nitrogen laser,
l=337 nm) were accelerated to a kinetic energy of 23.5 kV. Metastable
ions generated by laser-induced decomposition of the selected precur-
sor ions were analyzed without any additional collision gas. Precursor
ions were accelerated to 8 kV and selected in a timed ion gate. The
fragments were further accelerated by 19 kV in the LIFT cell, and their
masses were analyzed after the ion reflector passage. Masses were
automatically annotated by using XMASS 5.1.2 NT.
In conclusion, this study demonstrated that glycoblotting
based on oxylamino-containing polymers (Fischer-type poly-
mer reagents) could generally be used for capturing both
common oligosaccharides and aldehyde-attached glycopep-
tides derived by enzymatic modifications. It should be noted
that the efficiency and versatility in the capturing of
carbohydrates from the crude proteolytic digests is crucial
for ideal and practical protein glycomics. Therefore, the
combined use of glycoblotting and high-performance
MALDI-TOF/TOFanalysis provides us with a new promising
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Chemie
Figure 3. Identification of the ratio between isomers A and B found in the molecular ion peak at m/z 1648 by using carbohydratemass fing er print-
ing (CMF) analysis recorded in LIFT-TOF/TOF mode. The relative intensities of LIFT-TOF/TOF product ion peaks of unknown carbohydrate struc-
ture were subjected to matching simulation with the correlation coefficient analysis. Two known oligosaccharide derivatives were used for achiev-
ing thestandard CMF data.
Figure 4. Identification of the O-GlcNAc modified peptides by MALDI-TOF mass spectrometry. a) MALDI-TOF MS of the reaction mixture after
enzymatic galactosylation of glycopeptide, b) MALDI-TOF MS of an isolated glycopeptide derivative by glycoblotting method, and c) MALDI-LIFT-
TOF/TOF tandem mass analysis of the precursor ion peak at m/z 1473.71.
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strategy for early diagnoses and tailored treatments of a
variety of diseases, as well as high-throughput protein
glycomics.
Received: August 17, 2004
Keywords: carbohydrates · chemoselectivity · glycoproteins ·
.
high-throughput screening · mass spectrometry
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[
13] An aldehyde group generated at the C-8 position of the terminal
sialic acid of a glycoprotein can also be trapped by this method.
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4
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