Relative quantitation of glycopeptides based on stable isotope labeling using MALDI-TOF MS

Article abstract of DOI:10.3390/molecules19079944

We have developed an effective, sensitive method for quantitative glycopeptide profiling using stable isotope labeling and MALDI-TOF mass spectrometry (MS). In this study, we synthesized benzoic acid-d0 N-succinimidyl ester (BzOSu) and benzoic acid-d5 N-s

Full text of DOI:10.3390/molecules19079944

Molecules 2014, 19, 9944-9961; doi:10.3390/molecules19079944
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Relative Quantitation of Glycopeptides Based on Stable Isotope
Labeling Using MALDI-TOF MS
Masaki Kurogochi and Junko Amano *
Laboratory of Glycobiology, The Noguchi Institute, 1-8-1, kaga, Itabashi, Tokyo 173-0003, Japan;
E-Mail: kuroko@noguchi.or.jp
* Author to whom correspondence should be addressed; E-Mail: amano@noguchi.or.jp;
Tel.: +81-3-3961-3255; Fax: +81-3-3964-5588.
Received: 31 March 2014; in revised form: 1 July 2014 / Accepted: 7 July 2014 /
Published: 9 July 2014
Abstract: We have developed an effective, sensitive method for quantitative glycopeptide
profiling using stable isotope labeling and MALDI-TOF mass spectrometry (MS). In this
study, we synthesized benzoic acid-d0 N-succinimidyl ester (BzOSu) and benzoic acid-d5
N-succinimidyl ester (d-BzOSu) as light and heavy isotope reagents for stable isotope
quantification for the comparative analysis of glycopeptides. Using this approach provided
enhanced ionization efficiency in both positive and negative modes by MALDI-TOF MS.
These reagents were quantitatively reacted with glycopeptides from human serum IgG
(hIgG) at a wide range of concentrations; the labeling efficiency of the glycopeptides
showed high reproducibility and a good calibration curve was obtained. To demonstrate the
practical utility of this approach, we characterized the structures of glycopeptides from
hIgG and from IgG1 produced by myeloma plasma. The glycopeptides were quantitatively
analyzed by mixing Bz-labeled IgG1 glycopeptides with d-Bz-labeled hIgG glycopeptides.
Glycan structural identification of the hIgG glycopeptides was demonstrated by combining
the highly specific recognition of endo-β-N-acetyl glucosaminidases from Streptococcus
pyogenes (endoS) or from Streptococcus pneumoniae (endo-D) with MALDI-TOF MS
analysis. The obtained data revealed the glycan profile and the ratio of glycan structural
isomers containing a galactosylated extension on IgG1, IgG2 and IgG3 glycopetides.
Keywords: glycopeptides; stable isotope labeling; MALDI TOF-MS; quantitative
glycopeptide profiling

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1. Introduction
Glycosylation is one of the most ubiquitous and crucial processes in the posttranslational
modification of proteins expressed in eukaryotic cells [1,2]. It is estimated that 50%–60% of human
proteins are glycosylated [3]. The glycans play important roles in many biological phenomena, such as
the stability and conformation of the protein, cellular signaling, and molecular recognition.
Glycoproteomics combines proteomics and glycomics and aims to characterize the sequence,
glycosylation sites, and glycan structures of the glycosylated peptides and proteins, including their
microheterogeneity. Glycoproteomics has recently been used to elucidate biological functions and to
explore biomarkers. The study of glycoproteomics is complicated not only because of the variety of
carbohydrates that decorate proteins, but also because of the complex linkages of the glycan to the
protein. Glycosylation occurs at several different amino acid residues in the protein sequence. The
most common and widely studied forms are N-linked and O-linked glycosylation. N-linked glycans are
attached to the side chain amide group of asparagine residues in a consensus Asn-X-Ser/Thr sequence,
whereas O-linked glycans are linked to the side chain hydroxyl group of serine or threonine residues.
The techniques used in glycoproteomics are becoming more sophisticated, allowing more detailed
information to be obtained [4]. The development of these techniques has led to clinical translational
studies and its application to several fields, in particular to protein biomarker research. These
techniques are classified into three categories: glycoprotein and glycopeptide enrichment, systematic
identification, and the quantification of glycopeptides in a complex sample. Several methods have been
reported for glycoprotein and glycopeptide enrichment, such as lectin affinity approaches [5,6], hydrazide
chemistry-based solid phase extraction approaches [7–10], boronic acid affinity approaches [11,12], and
hydrophilic interaction liquid chromatography (HILIC) approaches [13,14]. Systematic identification
has benefited from developments in instrumentation such as high performance fragmentation mass
spectrometers (high energy collision-induced dissociation (HCD) [15], infrared multiphoton
dissociation (IRMPD) [16,17], electron-capture dissociation (ECD) [16–20], and electron-transfer
disassociation (ETD) [21,22]), as well as developments in search algorithms such as SEQUEST [23],
MASCOT [24], and X! tandem [25]. A large number of annotated glycoproteins have been recorded in
the UniProt database, and many MS/MS spectra are listed in the PeptideAtlas library [26]. For
quantitative glycoproteomics, stable isotopic or isobaric labeling using chemical reactions (ICAT and
iTRAQ) [27,28], metabolic incorporation (SILAC) [29], enzymatic reactions (18O labeling [30]) and
MRM-MS methods using ESI-triple-quadrupole MS instruments as label-free approaches [31] have
been developed. These techniques are generally based on LC-ESI-MS/MS systems with high
separation performance. However, LC-ESI-MS/MS instruments are more complicated to operate than
MALDI-TOF MS; for example, the ionization parameters (probe temperature, voltage, etc.) and LC
parameters must be adjusted so that the multiply charged ions can be identified. In MALDI-TOF MS,
stable isotope labeling of peptides and glycans has been reported [27,32,33], but these labeling
approaches are not suitable for glycopeptides because glycopeptides exhibit low reactivity and
ionization efficiency due to the large number of hydroxyl groups in carbohydrates. We previously
developed an on-target labeling method for glycopeptides with pyrenyl diazomethane to enhance
ionization [34]. The derivative is neutral, resulting in effective ionization and the production of
positive and negative ions. However, this methodology is not suitable for comparative analysis using

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stable isotope labeling because the derivative is prepared on-target. This work describes how we
developed a novel strategy based on stable isotope labeling allowing the highly sensitive relative
quantitation of glycopeptides. The labeling of glycopeptides has been applied to cation-charged mass
tags [35] bearing a quaternary phosphonium cation or a tertiary, quaternary ammonium cation, such as
tris-(2,4,6-trimethoxyphenyl)-phosphonium (TMPP) derivatives, and also to a tandem mass tag
(TMT) [36,37]. These approaches are similar to those used in proteomics and glycomics [38]. Stable
isotope dimethyl labeling to amino groups of peptides and glycopeptides by reductive amination was
often applied to LC-MS analysis [39–41], but it seems to be unsuitable for detection in negative mode
due to a formation of tertiary ammonium ion. Although we synthesized the benzoyl-d0 (Bz) and
benzoyl-d5 (d-Bz) reagents as non-charged hydrophobic mass tags, they enhanced ionization efficiency
in both the positive and negative modes in MALDI-TOF MS. To demonstrate the utility of the
developed approach, we characterized and quantitatively analyzed the N-linked glycopeptides of IgG1
from two samples: human serum IgG, and purified IgG1 produced by myeloma plasma. In addition,
we applied this method to identify the substrate specificity of endo-glycosidase and obtained glycan
structural identification using the highly specific recognition of endo-β-N-acetylglucosaminidases
(endoS and endo-D). Identification was achieved using MALDI-TOF MS on crude samples. This study
suggests that isotopic glycopeptide-labeling is a promising approach for quantitative glycopeptide
profiling and holds significant potential for biological and clinical research.
2. Results and Discussion
2.1. Synthesis and Evaluation of Stable Isotope-Labeled Amine-Reactive Mass Tag Reagent
for Glycopeptides
Stable isotope labeling mass tags have been exploited for both the relative and absolute
quantification of peptides in proteomic studies [27,28,36,37].
We synthesized benzoic acid-d0 N-succinimidyl ester (BzOSu) and benzoic acid-d5 N-succinimidyl
ester (d-BzOSu) as light and heavy isotope mass tag reagents for stable isotope-based quantification in
comparative glycoproteomics (Scheme 1). These compounds were functionalized with amine-reactive
groups, and reacted with amine-containing glycopeptides to form an amide bond through a mild
coupling reaction. The coupled glycopeptides could be detected under both positive and negative mode
conditions in MALDI-TOF MS due to their enhanced MS signal compared to unlabeled glycopeptides.
Enhanced MS signals were also observed for egg yolk glycopeptides and bovine ribonuclease B
glycopeptides, in a manner similar to that for hIgG glycopeptides (Supplementary Figures S1–S3). We
thought that a non-charged hydrophobic mass tag would enhance ionization efficiency by preventing
the aggregation of analytes, while ionic interactions and hydrophilic interactions would induce
aggregation and inhibit the ionization process in both positive and negative modes on MALDI-TOF
MS. Although we tested other labeling compounds, including the acetyl, naphthoyl and pyrenoyl
groups, with egg yolk glycopeptides, comparative analysis by MALDI-TOF MS (Supplementary
Figures S1, S4 and S5) showed that the benzoyl group was the best labeling compound for enhancing
ionization efficiency. The hydrophobicity of the benzoyl group may be sufficient to inhibit the
hydrophilic interaction of glycopeptides, without causing hydrophobic aggregation. We selected the

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benzoyl group to label glycopeptides and quantified the labeled glycopeptides by their MS spectra
using isotopic light/heavy pairs (Figure 1).
Scheme 1. Synthesis of d0-BzOSu (A) and d5-BzOSu (B).
Reagents: HOSu: N-hydroxysuccinimide; TEA: triethylamine; DCC: dicyclohexylcarbodiimide;
DMF: dimethylformamide).
Figure 1. Strategy for quantitative and comparative analysis based on stable isotope
labeling using MALDI TOF-MS.
The mass difference of 5.03 Da between the light and heavy benzoyl reagents was sufficient to
separate the isotopic patterns of the glycopeptides. Although N-hydroxysuccinimide ester reacts with
all amino groups of peptides containing the side chain ε-amino group of a lysine residue, this reagent
can be incorporated at only N-terminal amino group of peptides after blocking the side chain ε-amino

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group of a lysine residue with guanidination in the same procedure as Chengjie et al.’s [40], for
example Supplementary Figures S1,S3.
Figure 2. Validation of the proposed method using glycopeptides from human serum IgG.
(A) MALDI-TOF MS spectra of (i) Bz-labeled glycopeptides (ii) an equimolar mixture of
Bz- and d-Bz-labeled glycopeptides (iii) d-Bz labeled glycopeptides; (B) Enlarged spectra
of glycopeptides containing Hex3HexNAc3dHex1, Hex4HexNAc4dHex1, Hex4HexNAc5dHex1;
(C) Plots of each peak in the equimolar mixture of Bz- and d-Bz-labeled glycopeptides.
(red, blue is IgG1, IgG2/3 glycopeptides; circle, square is Bz-, d-Bz-labeled glycopeptides).

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Glycopeptides were prepared from normal hIgG and reacted with light and heavy isotope mass tag
reagents. The labeled glycopeptides were isolated from the complex sample using the improved HILIC
method reported by Wada et al as described in Experimental Section 3.4 [13]. Then, each obtained
isotopically labeled glycopeptides and a 1:1 molar ratio mixture were measured by MALDI-TOF MS,
we observed labeled glycopeptides with the mass difference of 5 Da origined from light and heavy
isotope mass tag (Figure 2).
Human serum IgG is composed of four subclasses (IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4
(4%)); the characteristics of these subclasses have been reported extensively [42–44]. All four
subclasses have a single N-glycosylation site in the constant region of the heavy chain. This single
N-glycosylation site was occupied by partially truncated biantennary glycans with or without core
fucosylation or bisecting GlcNAc. Glycopeptides digested with trypsin provided four peptide moieties
consisting of nine amino acids. These peptides were derived from the constant region of the heavy
chains of IgG1, IgG2, IgG3 and IgG4; consequently, these glycopeptides could be identified by the
peptide molecular weight. In this study, we observed the isotopically labeled glycopeptides consisting of
EEQYNSTYR (1189.2 Da) from IgG1 and EEQFNSTFR (1157.2 Da) from IgG2/3 (IgG2 and IgG3
were abbreviated as IgG2/3) (Figure 2). However, glycopeptides with the sequence EEQYNSTFR
(1173.2 Da) from IgG3 and EEQFNSTYR (1173.2 Da) from IgG4 were not detected due to their low
concentration. The glycopeptides from IgG1 and IgG2/3 could be quantitatively reacted with isotopic
reagent, and the obtained glycopeptides by HILIC purification could be quantitatively isolated from
mixture for the reason that unglycosylated peptides were not mainly detected (Figure 2A), but Bz- and
dBz-labeled glycopeptides with the same peak intensities were observed in the spectra of an equimolar
mixture (Figure 2B). These glycopeptides had 10 glycoforms exhibiting a large dynamic range (about
100-fold) with concentration (Supplementary Table S1). Although the glycopeptides of human serum
IgG have a small amount of sialic acid [43], loss of sialic acid occurs due to post-source decay during
MALDI-TOF MS measurement in positive mode (Supplementary Figures S10). This work was
focused on neutral glycopeptides without sialic acid. Also, the results showed that the glycan profiles
between IgG1 and IgG2/3 were different (Figure 2C). In MALDI-TOF MS, the MS signal ratio of
IgG1 glycopeptides and IgG2/3 glycopeptides depended on the amounts of analytes, but the MS signal
ratio of the glycan profile on the same peptide sequence was retained regardless of the concentration of
the analytes (Supplementary Figure S6). The difference in ionization efficiency induced by
stabilization of the coordination complex (proton adduct ion affected by the proton affinity of the
molecules) depended on the concentration and characteristics affected by composition of the
molecules. Consequently, this stable isotope labeling of glycopeptides was essential to quantitative
glycopeptide profiling by MALDI-TOF MS, which could be used as measurement standard having the
same composition and concentration.
2.2. Calibration of IgG1 Glycopeptides Using Stable Isotope Labeling
To examine the accuracy and reproducibility of glycopeptide quantification using isotopic labeling,
we labeled glycopeptides from purified IgG1 with light (Bz) and heavy (d-Bz) reagents, mixed them in
different molar ratios (1:0.43, 1:0.71, 1:1, 1:1.29, 1:1.57, 1:1.86, 1:2.14, Bz- to d-Bz-labeled glyco-
peptides), and analyzed the mixtures by MALDI-TOF MS (Figure 3). The obtained IgG1 glycopeptides

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by HILIC purification could be quantitatively isolated from mixture in the same way as human serum
IgG experiment. As shown in the spectra of mixture (Figure 3A,B), these spectra exhibited five pairs
of peaks, including those with m/z 2737.8/2742.8, 2899.8/2904.8, 2940.8/2945.9, 3061.9/3066.9 and
3102.9/3107.9.
Figure 3. (A) MALDI-TOF MS spectra of Bz and d-Bz labeled glycopeptides mixed in
different molar ratios (1:0, 1:0.43, 1:0.71, 1:1, 1:1.29, 1:1.57, 1:1.86, 1:2.14, 0:1); (B)
Enlarged spectra of IgG1 glycopeptides containing Hex3HexNAc4dHex1, Hex4HexNAc4dHex1,
Hex3HexNAc5dHex1, Hex5HexNAc4dHex1, Hex4HexNAc5dHex1; (C) Calibration curves
(peak intensity ratio vs. H/D molar ratio, H/D ratio means Bz-/d-Bz-labeled glycopeptides).
These pairs were assigned to the Bz- and d-Bz-labeled glycopeptides containing
Hex3HexNAc4dHex1, Hex4HexNAc4dHex1, Hex3HexNAc5dHex1, Hex5HexNAc4dHex1 and
Hex4HexNAc5dHex1, respectively. The MS signal intensity ratios of Bz- to d-Bz-labeled glycopeptides
were plotted versus the expected molar ratios (Figure 3C). The response curves were not quite linear
and were fit using power approximation by the approach reported by Anderson et al. [45] to provide the

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R2 value and coefficient of variability (CV) of the mean value (n = 3) (Figure 3C). Although the
reproducibility of components (m/z 2940.8/2945.9 and 3061.9/3066.9) with high CV values present at
low concentration was worse than that of components present at high concentration, a resulting
response curve of a component was almost overlapped with other component’s response curve
(Supplementary Figure S9). Thus, the calibration curves can be used to calculate the concentration of
targeted molecules, and also to characterize the glycoform contents.
2.3. Quantitative Comparison of Glycopeptides from Human Serum IgG and IgG1 κ from Myeloma Plasma
To validate the applicability of the newly developed method to the quantitative glycopeptide
profiling of two samples, the relative quantification between IgG1 subclass from human serum IgG
and purified IgG1 from human myeloma plasma was performed. The two samples were prepared and
labeled with d-BzOSu and BzOSu as described in the experimental Sections 3.2 and 3.3. The
isotopically labeled glycopeptide samples were then mixed in various ratios and analyzed by
MALDI-TOF MS (Figure 4).
Figure 4. Comparative analysis between IgG1 and hIgG. (A) MALDI-TOF MS spectra of
different ratio mixtures (0:2.50, 1.60:2.50, 1.60:3.33, 1.60:5.00, 1.60:7.14, 1.60:10.0, 1.60:0
(μg/μg)) of Bz-labeled IgG1 to d-Bz-labeled hIgG glycopeptides; (B) Enlarged spectra of IgG1
glycopeptides containing Hex3HexNAc4dHex1, Hex4HexNAc4dHex1, Hex5HexNAc4dHex1;
(C) The plots of each glycopeptide ratio of the two glycoproteins.

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Figure 4. Cont.
The different ratio mixtures (0:2.50, 1.60:2.50, 1.60:3.33, 1.60:5.00, 1.60:7.14, 1.60:10.0, 1.60:0 of
Bz-labeled IgG1 to d-Bz-labeled hIgG glycopeptides derived from original amounts of protein based
on weight (μg)) produced three pairs of peaks. Three pairs were the glycopeptides consisting of three
kinds of glycan composition (Hex3HexNAc4dHex1, Hex4HexNAc4dHex1 and Hex5HexNAc4dHex1)
and the same amino acid sequence (EEQYNSTYR), so that human serum IgG1 glycopeptides could be
identified from the spectra of human serum IgG mixture. The MS signal intensity ratio of each labeled
glycopeptide to the total labeled IgG1 glycopeptides containing three kinds of glycans with the same
isotope mass tag was retained at each mixture, and also this strategy indicated high accuracy and
reproducibility for glycopeptides profiling containing the same amino acid sequence. A difference of
glycan profiling between IgG1 among human serum IgG and purified IgG1 was shown by this method
(Figure 4C). In addition, the ratio of IgG1 to human serum IgG was approximately 64% (w/w) under
the calculation that total amount of IgG1 glycopeptides obtained from 5.00 μg of human serum IgG was
twice as much as total amount glycopeptides from 1.60 μg of purified IgG1. This approach showed that
isotopic quantitative glycopeptide profiling leads to characterization of glycan profiling and relative
quantitation of targeted glycoprotein and glycopeptides in the mixture of biological samples.
2.4. Quantitative Monitoring of Glycopeptides for Enzymatic Reaction using Stable Isotope Labeling
2.4.1. Substrate Specificity of Endo-β-N-acetylglucosaminidase from Streptococcus Pyogenes (endoS)
for hIgG Glycopeptides
To explore the substrate specificity of endo-β-N-acetylglucosaminidase from Streptococcus
pyogenes (endoS) [46], quantitative comparison of the glycopeptides with and without enzymatic
reaction was performed. hIgG glycopeptides labeled with BzOSu and d-BzOSu were prepared as
described in the experimental Sections 3.3 and 3.4, then the Bz-labeled glycopeptides were reacted
with endoS, and mixed with d-Bz-labeled peptides in one of four ways: (i) Bz-labeled hIgG
glycopeptides reacted with endoS; (ii) Bz-labeled hIgG glycopeptides reacted with endoS and d-Bz-
labeled hIgG glycopeptides (1:1 ratio) without reaction of endoS; (iii) Bz-labeled hIgG glycopeptides

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and d-Bz-labeled hIgG glycopeptides in a 1:1 ratio; (iv) d-Bz-labeled hIgG glycopeptides. From
MALDI-TOF MS analysis of these samples (Figure 5), it was confirmed that Bz-labeled hIgG
glycopeptides and d-Bz-labeled hIgG glycopeptides with the same concentration as an enzymatic
substrate could be precisely prepared, and also enhanced ionization potency of labeled glycopeptides
allowed for MALDI-TOF MS based facile and quantitative monitoring of enzymatic reaction. The
glycopeptides (m/z 1464.3 for IgG2/3, 1496.3 for IgG1, and 1610.4 for IgG2/3, 1642.4 for IgG1)
bearing a monosaccharide (GlcNAc) or a disaccharide (Fucα1-6GlcNAc) prominently appeared after
reaction with endoS (Figure 5A): most of the hIgG glycopeptides were digested by the reaction of
endoS. However, glycopeptides containing bisecting N-glycans, i.e., containing a GlcNAcβ1-4 linked
β-mannose residue in the N-glycan core structure, were not digested (Figure 5B). Although Dixon E.V.
et al. recently reported the susceptibility of endoS for IgG and IgG fragments (Fc and CH2-H, CH2
domain), they did not examine trypsin-digested glycopeptides. After the reaction of endoS, there was a
population of uncleaved bisecting biantennary glycans from the CH2-H and CH2 glycoprotein except
for IgG and Fc glycoprotein which can be recognized by carbohydrate binding modules (CBM) on
endoS. Thus, the result is consistent with our result that endoS is hard to cleave biantennary
N-glycans containing bisecting GlcNAc in comparison with other biantennary N-glycans on IgG
glycopeptides [46,47].
Figure 5. (A) MALDI-TOF MS spectra of (i) Bz-labeled hIgG glycopeptides reacted with
endoS (ii) equimolar mixture of Bz-labeled hIgG glycopeptides reacted with endoS and
d-Bz-labeled hIgG glycopeptides not reacted with endoS (iii) equimolar mixture of
Bz-labeled hIgG glycopeptides and d-Bz-labeled hIgG glycopeptides (iv) d-Bz-labeled
hIgG glycopeptides; (B) Enlarged spectra from m/z 2400 to 3400; (C) Enlarged spectra of
IgG1 glycopeptides containing Hex4HexNAc5dHex1.

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2.4.2. Characterization of the hIgG Glycopeptide Isoforms by endo-β-N-Acetylglucosaminidase from
Streptococcus pneumoniae (endo-D) Combined with exo-β-N-Acetylglucosaminidase (β-GlcNAc’ase)
To characterize the glycan structural isomers of the hIgG glycopeptides by MALDI-TOF MS
analysis, quantitative comparison of glycopeptides was performed using the known substrate
specificity of exo-β1–2,3,4,6-N-acetylglucosaminidase (β-GlcNAc’ase) and endo-β-N-acetyl-
glucosaminidase from Streptococcus pneumoniae (endo-D). hIgG glycopeptides labeled with BzOSu
and d-BzOSu were prepared and digested with β-GlcNAc’ase as described in the experimental
Section 3.6. Both Bz- and d-Bz-labeled glycopeptides from hIgG were converted to glycopeptides with
an unsubstituted α-mannosyl residue at the terminal mannose of the N-glycan core structure in part,
and then these labeled glycopeptides were prepared as a mixture containing the upper branch and the
lower branch with galactosylated extension on the biantennary glycan. The N-glycans from normal
hIgG have previously been identified by HPLC separation [48,49]. The molar ratio of the upper to the
lower galactosylated extension on partially truncated biantennary glycans with core fucosylation was
shown to be about 2.5:1.0. Generally, glycan structural isomers with the same molecular weight cannot
be determined by mass spectrometry, but can be recognized by a specific protein (lectin) or enzyme
exhibiting high affinity and substrate specificity. In this experiment, quantitative characterization of
glycan isomers on glycopeptide profiling was accomplished using stable isotope labeling and the high
substrate specificity of endo-D. Endo-D cleaves only N-glycans with an unsubstituted α-mannosyl residue
at the C-3 position of the terminal mannose of the N-glycan core structure from glycopeptides [50]. These
two approaches allowed determination of the glycan structural isomer. hIgG glycopeptides labeled
with BzOSu were reacted with β-GlcNAc’ase and then with endo-D, and then mixed with d-Bz-
labeled hIgG in one of two ways: (i) Bz-labeled hIgG glycopeptides were reacted with both
β-GlcNAc’ase and endo-D, whereas d-Bz-labeled hIgG glycopeptides were reacted with only
β-GlcNAc’ase; (ii) Bz-labeled hIgG glycopeptides were reacted with only β-GlcNAc’ase and d-Bz
labeled hIgG glycopeptides were reacted with only β-GlcNAc’ase. It was confirmed that Bz-labeled
hIgG glycopeptides and d-Bz-labeled hIgG glycopeptides with the same concentration was precisely
prepared, and then enhanced ionization of labeled glycopeptides allowed for MALDI-TOF MS based
facile and quantitative characterization of glycan isomers on glycopeptides (Figure 6). The
glycopeptides (m/z 1464.3 for IgG2/3, 1496.3 for IgG1, and 1610.4 for IgG2/3, 1642.4 for IgG1)
bearing a monosaccharide (GlcNAc) or a disaccharide (Fucα1-6GlcNAc) appeared after reaction with
endo-D (Figure 6A). The MS signals of these digested glycopeptides were prominent because most of
the glycopeptides had reacted with endo-D. All hIgG glycopeptides (m/z 2299.6 for IgG2/3, 2331.6 for
IgG1 consisting of Hex3HexNAc2dHex1) with unsubstituted α-mannosyl residues in the trimannosyl
N-glycan core structure were completely digested with endo-D, but glycopeptides (m/z 3029.7 for
IgG2/3, 3061.7 for IgG1 consisting of Hex5HexNAc4dHex1) containing fully substituted α-mannosyl
residues remained undigested. In the case of glycopeptides (m/z 2664.7 for IgG2/3, 2696.7 for IgG1
consisting of Hex4HexNAc3dHex1) containing partially substituted α-mannosyl residues, 40% and
20% of the MS signal remained, respectively (Figure 6B). Calculations based on the calibration curves
for the target molecules (Supplementary Figure S9) showed that the molar ratios of the upper to lower
galactosylated extension were 81:19 for IgG1 glycopeptides and 57:43 for IgG2/3 glycopeptides
(Figure 6C). Finally, it was found that the ratio of the N-glycan structural isomer containing the

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galactosylated extension was different between IgG1 and IgG2/3 from human serum IgG.
Furthermore, isotopic quantitative glycopeptide profiling by MALDI-TOF MS was useful for
characterizing glycan structural isomers by combination with the highly specific recognition of an
enzyme such as endo-D.
Figure 6. (A) MALDI-TOF MS spectra of (i) an equimolar mixture of Bz-labeled hIgG
glycopeptides reacted with both β-GlcNAc’ase and endo-D, and d-Bz-labeled hIgG
glycopeptides reacted with only β-GlcNAc’ase (ii) an equimolar mixture of Bz-labeled
hIgG glycopeptides reacted with only β-GlcNAc’ase and d-Bz-labeled hIgG glycopeptides
reacted with only β-GlcNAc’ase; (B) Enlarged spectra of glycopeptides containing
Hex3HexNAc2dHex1, Hex4HexNAc3dHex1, Hex5HexNAc4dHex1; (C) The molar ratio of
glycoforms on the IgG1 and IgG2/3 glycopeptides.
3. Experimental Section
3.1. General Information
The 1H-, 13C-NMR spectra were obtained on a JEOL JNM ECA instrument (JEOL, Tokyo, JAPAN)
at 600 MHz for 1H-NMR and 150 MHz for 13C-NMR. Deuterium-labeled benzoic acid was purchased
from Cambridge Isotope Laboratories (Andover, MA, USA), and benzoic acid was purchased from
Tokyo Chemical Industry (Tokyo, Japan). Human serum immunoglobulin G (hIgG), IgG1, kappa from
human myeloma plasma and Sepharose 4B were purchased from Sigma-Aldrich (Milwaukee, WI,
USA). RapiGest SF was purchased from Waters (Milford, MA, USA). TPCK trypsin was purchased
from Thermo Fisher Scientific (Rockford, IL, USA). Sephadex G-25 was purchased from GE

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Healthcare Life Sciences (Uppsala, Sweden). Endo-β-N-acetylglucosaminidase from Streptococcus
pyogenes (endoS) was overproduced in E. coli using a previously reported procedure [46]. Endo-β-N-
acetylglucosaminidase from Streptococcus pneumoniae (endo-D) was purchased from New England
Biolabs (Ipswich, MA, USA). β1–2,3,4,6-N-Acetylglucosaminidase from Streptococcus pneumoniae
was purchased from Merck KGaA (Darmstadt, Germany). Other chemicals were purchased from
Wako Pure Chemical Industries (Osaka, Japan).
3.2. Synthesis of Benzoic Acid N-Succinimidyl Ester (Bz and d-Bz Labeling Reagents)
Benzoic acid (ring d-5, heavy isotope, d-Bz) (127 mg, 1.00 mmol) or benzoic acid (ring d-0,
light isotope, Bz) (122 mg, 1.00 mmol), N-hydroxysuccinimide (230 mg, 2.00 mmol) and
triethylamine (167 μL, 1.20 mmol) were dissolved in anhydrous dimethylformamide (DMF) (1 mL).
After addition of dicyclohexylcarbodiimide (DCC) (206 mg, 1.20 mmol), the solution was stirred at
room temperature for 3 h. The reaction mixture was filtered to remove the precipitate, and
concentrated in vacuo. The resulting syrup was crystallized from isopropanol (5 mL) to yield benzoic
acid N-succinimidyl ester (d-Bz labeling reagent, 184 mg, 82%) (Bz labeling reagent, 170 mg, 78%).
Rf = 0.42 (toluene-EtOAc = 10:1); 1H-NMR (600 MHz, CDCl3): δ 2.91 (s, 4H) for the heavy isotope, δ
13
2.91 (s, 4H), 7.52 (dd, 2H), 7.69 (dd, 1H), 8.14 (dd, 2H) for the light isotope; C-NMR (125 MHz,
CDCl3): δ 25.63, 124.88, 128.31, 130.13, 134.38, 161.82, 169.24 for the heavy isotope, δ 25.65,
125.08, 128.83, 130.55, 134.91, 161.84, 169.23 for the light isotope.
3.3. Preparation of hIgG and IgG1 Glycopeptides
Human IgG (200 μg) or IgG1 (200 μg) was dissolved in 500 μL of 50 mM ammonium bicarbonate
solution and heated at 100 °C for 15 min. After the sample cooled, 50 μL of 1% aqueous RapiGest SF (v/v)
and 2 μL of TPCK-treated trypsin (20 μg) were added, followed by incubation at 37 °C for 300 min. The
sample was heated at 100 °C for 15 min to inactivate the enzyme, desalted by passage through a G-25
gel filtration column (0.8 × 3.5 cm), and concentrated using a centrifugal evaporation system.
3.4. Stable Isotope Labeling of Glycopeptides
The glycopeptides were dissolved in 20 μL of water, then 10 μL of pyridine and 20 μL of 200 mM
benzoic acid N-succinimidyl ester solution (d-Bz or Bz isotope labeling reagent) in DMF was added
and reacted at 57 °C for 12 h. After addition of 60 μL of 0.5 M NaOHaq to de-esterify the product, the
sample was mixed on a vortex mixer at room temperature for 30 min. Water (200 μL) was added, the
sample was washed with EtOAc (400 μL × 3 times) to remove excess reagent, and the aqueous layer
was collected and concentrated using a centrifugal evaporation system. The sample was desalted on a
C-18 Spin column (20 mg of C-18 reverse phase silica gel) and concentrated using a centrifugal
evaporation system. Further purification was done by HILIC method, which was modified Wada’s [13].
The sample was dissolved in 20 μL of water, added to a mixture of Sepharose 4B (wet vol. 50 μL),
100 μL of ethanol and 400 μL of butanol, then mixed on a tube rotator at room temperature for 1 h.
The resin was washed thoroughly with 2 ml of 10:2.5:2.2:0.3 (v/v/v/v) butanol/ethanol/water/formic

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acid to remove unglycosylated peptides, then the labeled glycopeptides were eluted with 1.3 mL of
25% ethanol and concentrated using a centrifugal evaporation system.
3.5. Enzymatic Reaction of Bz-Labeled Glycopeptides with EndoS
The Bz-labeled glycopeptides obtained from 50 μg of hIgG were dissolved in 5 μL of 10 mM
sodium phosphate buffer (pH 7.4) and incubated with endoS (0.5 μL, 1.0 μg/μL) at 37 °C for 12 h. The
sample was directly analyzed by MALDI-TOF MS without purification.
3.6. Enzymatic Reaction of Labeled Glycopeptides by β-N-Acetylglucosaminidase (β-GlcNAc’ase)
The Bz- or d-Bz-labeled glycopeptides obtained from 100 μg of hIgG were dissolved in 10 μL of
50 mM sodium acetate buffer (pH 5.0) and incubated with β-GlcNAc’ase (50 mU, 2 μL) at 37 °C for
12 h. After heating at 100 °C for 15 min, the sample was desalted on a C-18 Spin column (20 mg of
C-18 resin) and concentrated using a centrifugal evaporation system.
3.7. Enzymatic Reaction of Bz-Labeled Glycopeptides by Endo-D
The hydrolyzed Bz-labeled glycopeptides obtained from 50 μg of hIgG were dissolved in 5 μL of
10 mM sodium phosphate buffer (pH 7.4), and incubated with endo-D (0.5 μL, 25 units) at 37 °C for
12 h. The sample was directly analyzed with MALDI- TOF MS without purification.
3.8. MALDI Sample Preparation and Measurement
The sample solution (0.5~1.0 μL) was mixed with 1 μL of matrix (10 mg/mL 2,5-dihydroxybenzoic
acid solution [Shimadzu Biotech, Kyoto, Japan]) on a target plate. Mass spectra were acquired in
positive ion mode using a matrix assisted laser desorption ionization quadrupole ion trap time of flight
mass spectrometer (MALDI-QIT-TOF MS) (AXIMA Resonance, Shimadzu Biotech, Manchester,
UK). Ions were generated by a pulsed nitrogen UV laser (337 nm, 5 Hz).
4. Conclusions
A novel strategy based on stable isotope labeling with BzOSu and d-BzOSu allows the highly
sensitive relative quantitation of glycopeptides. To our knowledge, this approach is the first
demonstration of isotopic reagents for glycopeptide labeling allowing comparative glycopeptide
profiling studies by MALDI-TOF MS. We synthesized Bz and d-Bz reagents, determined the
optimized reaction conditions for Bz and d-Bz labeling, and validated the detection sensitivity of
benzoyl labeling using egg yolk glycopeptides, human IgG, and bovine ribonuclease B glycopeptides
as model glycopeptides. The sensitivity of detection of the glycopeptides was approximately 2 fmol
(Supplementary Figure S6). The high reproducibility and accuracy of the method was confirmed using
glycopeptides digested from hIgG. Two examples were used to demonstrate the applicability of the
procedure: glycopeptides digested from human serum IgG and purified myeloma plasma IgG1 were
quantitatively compared by MALDI-TOF MS analysis, and the glycopeptides from hIgG were
compared with and without enzymatic treatment. This labeling approach has two important advantages
including (i) higher sensitivity than non-labeled glycopeptides in both positive and negative modes

Molecules 2014, 19
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(Supplementary Figures S1–S3); and (ii) MS/MS profiles arising from X-, Y-, A-, B-type
fragmentation enable rapid structural identification in both positive and negative modes
(Supplementary Figure S8). Although this study focused on the analysis of N-linked glycopeptides, the
strategy can be directly applied to other types of glycopeptides, such as O-linked glycopeptides. In
addition to Bz and d-Bz labeling, other isotopic reagents are compatible with quantitative
glycoproteomics strategies. Furthermore, isotopic labeling of glycopeptides mixtures derived from
biological samples such as serum will be applicable to LC-MALDI TOF MS and LC-ESI-MS analysis,
allowing high separation performance. This strategy therefore represents a facile and versatile
analytical methodology for comparative glycoproteomics and holds promise for biological analyses.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/7/9944/s1.
Acknowledgments
This work was supported partly by a grant from JSPS KAKENHI, Grant Number 23710246.
We thank M. Mori of The Noguchi Institute for her kind gift of endoS.
Author Contributions
M. Kurogochi and J. Amano designed the study. M. Kurogochi performed the experiments, and
wrote the paper. All authors reviewed and edited the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Not available.
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