Hydrophobic derivatization of N-linked glycans for increased ion abundance in electrospray ionization mass spectrometry

Article abstract of DOI:10.1007/s13361-011-0140-x

A library of neutral, hydrophobic reagents was synthesized for use as derivatizing agents in order to increase the ion abundance of N-linked glycans in electrospray ionization mass spectrometry (ESI MS). The glycans are derivatized via hydrazone formation and are shown to increase the ion abundance of a glycan standard more than 4-fold. Additionally, the data show that the systematic addition of hydrophobic surface area to the reagent increases the glycan ion abundance, a property that can be further exploited in the analysis of glycans. The results of this study will direct the future synthesis of hydrophobic reagents for glycan analysis using the correlation between hydrophobicity and theoretical non-polar surface area calculation to facilitate the development of an optimum tag for glycan derivatization. The compatibility and advantages of this method are demonstrated by cleaving and derivatizing N-linked glycans from human plasma proteins. The ESI-MS signal for the tagged glycans are shown to be significantly more abundant, and the detection of negatively charged sialylated glycans is enhanced.

Full text of DOI:10.1007/s13361-011-0140-x

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2011) 22:1309Y1317
DOI: 10.1007/s13361-011-0140-x
FOCUS: INTERDISCIPLINARY BIOLOGICAL MS: RESEARCH ARTICLE
Hydrophobic Derivatization of N-linked Glycans
for Increased Ion Abundance in Electrospray
Ionization Mass Spectrometry
1
2
1
1
S. Hunter Walker, Laura M. Lilley, Monica F. Enamorado, Daniel L. Comins,
1
David C. Muddiman
1
W. M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh,
NC 27695, USA
Department of Chemistry, Warren Wilson College, Asheville, NC 28815, USA
2
Abstract
A library of neutral, hydrophobic reagents was synthesized for use as derivatizing agents in
order to increase the ion abundance of N-linked glycans in electrospray ionization mass
spectrometry (ESI MS). The glycans are derivatized via hydrazone formation and are shown to
increase the ion abundance of a glycan standard more than 4-fold. Additionally, the data show
that the systematic addition of hydrophobic surface area to the reagent increases the glycan ion
abundance, a property that can be further exploited in the analysis of glycans. The results of this
study will direct the future synthesis of hydrophobic reagents for glycan analysis using the
correlation between hydrophobicity and theoretical non-polar surface area calculation to
facilitate the development of an optimum tag for glycan derivatization. The compatibility and
advantages of this method are demonstrated by cleaving and derivatizing N-linked glycans from
human plasma proteins. The ESI-MS signal for the tagged glycans are shown to be significantly
more abundant, and the detection of negatively charged sialylated glycans is enhanced.
Key words: Hydrophobic Derivatization, HILIC, nanoESI, N-Linked Glycans, Hydrazone
Formation
chemistry step in sample preparation, the benefits have been
shown to outweigh the time and cost; a 92000-fold
Introduction
he inherent hydrophobic bias in electrospray ionization
(ESI) has been exploited in numerous studies first for
nucleic acids [1], then peptides and proteins [2–7], and most
recently glycans [8–14] in order to increase the ion
abundance of analytes in mass spectrometry (MS). While
these techniques frequently introduce an additional wet
improvement in peptide signal has been reported [7]. The
addition of hydrophobic functionality to molecules via
chemical derivatization will not only increase the ion
abundance in ESI but can also have other beneficial aspects,
such as the ability to incorporate stable isotopes for
quantification [10, 15] and the enhanced information
acquired from fragmentation spectra in the permethylation
of glycans [16]. Analysis of glycans by ESI has been
hampered by the hydrophilicity of the sugar functional
groups causing them to have a higher free energy of
solvation and be more difficult to desorb from the electro-
spray droplet in the generation of gas-phase ions. Thus,
T
Electronic supplementary material The online version of this article
doi:10.1007/s13361-011-0140-x) contains supplementary material, which
is available to authorized users.
(
Correspondence to: David C. Muddiman; e-mail: david_muddiman@ncsu.edu
Received: 3 November 2010
Revised: 8 February 2011
Accepted: 15 March 2011
Published online: 3 May 2011

1
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S.H. Walker, et. al.: Library of Hydrophobic Hydrazide Reagents
derivatization using hydrophobic reagents can be used to
counter this obstacle. Imparting additional hydrophobic
functions onto glycan molecules allows the glycans to be
less solvated and have a higher surface activity in the
precursor electrospray droplet. As this droplet is desolvated
on the way to the MS, a series of Coulombic fission events
occur where a number of smaller progeny droplets (esti-
mated to be ~20 [17]) are ejected from the surface of the
original droplet [17–19]. In comparison with the native
glycans, the more hydrophobic, derivatized glycans are more
likely to have a higher surface activity and are significantly
enriched in the progeny droplets [20]. It is these progeny
droplets that are further desolvated and eventually produce
gas-phase ions [21], creating the hydrophobic bias in ESI.
Permethylation [22–24] and peracetylation [23, 25]
represent two initial methods used to derivatize glycans for
enhanced MS analysis. These methods have been shown to
enhance fragmentation spectra [26, 27] and increase the
glycan ion abundance. However, permethylation and per-
acetylation convert all hydroxyl, amino, and carboxylic acid
groups to their methyl- and acetyl-ether functions, respec-
tively and, thus, the m/z shift due to the derivatization is
variable for different sized glycans. Additionally, 100%
conversion has proven difficult and is variable for different
N-linked glycans. For example, due to the numerous
hydroxyl, amino, and carboxylic groups present in oligosac-
charides (~5 per monosaccharide and 7–18 monosaccharides
per N-linked glycan), a 0.1% change in the permethylation
efficiency can result in a 3%–10% efficiency difference
depending on the type and size of the glycan. In contrast, the
reducing terminus is a convenient location for derivatization
due to the availability of a free aldehyde group after the
enzymatic cleavage of N-linked glycans from proteins using
peptide: N-glycosidase F (PNGase F). Moreover, stable isotope
labels with a small, fixed mass shift can be incorporated into
the reagents while simultaneously increasing the hydrophobic-
efficiency can be routinely achieved using hydrazone
formation as a means of hydrophobic tagging of glycans
[13]. Danzylhydrazine [35] was the first of many reagents to
be coupled to glycans via hydrazone formation for enhanced
fluorescence or UV detection [36–38]. More recently,
hydrazone formation has been used for enhanced MS
detection [8, 12, 13], though an abundance of these reagents
is not readily available. Several different hydrazide reagents
have been developed and used to increase glycan abundance
[8, 12, 13]. Girard’s T reagent was first used to increase
mass spectral detection in order to incorporate a permanent
cationic charge onto the glycan [12]. However, recent
studies have shown decreases in ion abundance when
coupling Girard’s T reagent to glycans in nanoLC MS [8],
and it has been shown that neutral reagents are significantly
better than preformed cations when coupled to N-linked
glycans [8, 13].
Herein, a small library of hydrazide reagents has been
synthesized and is used to derivatize glycans in order to
determine the type of hydrophobic structure that most
effectively increases the ion abundance of glycans in ESI-
MS. The reagents studied are exclusively neutral compounds
due to the compatibility in positive and negative mode MS
and due to recent studies showing decreased electrospray
response when incorporating a permanent charge on the
reagent (vide supra) [13]. Additionally, the non-polar surface
areas (NPSA) for all the reagents have been calculated in
order to give a theoretical estimate of the hydrophobicity of
the reagents as it has been shown that the NPSA correlates
directly with the hydrophobicity of the analytes in peptide
analysis [5, 6]. Furthermore, the compatibility of the
hydrazone reagents with complex mixtures and their
inherent advantages are demonstrated by the derivatization
of N-linked glycans found in human plasma.
ity. Reductive amination is a prominent technique capable of Experimental
reacting with the reducing terminus of N-linked glycans and
Materials
has been used to enhance several analytical techniques
including UV and fluorescence detection [28–31] and hydro-
phobic derivatization [9, 10, 32, 33]. Two studies have shown
the ability to incorporate hydrophobic tagging and isotopic
labeling using reductive amination derivatization [10, 15].
Though this method is effective, an additional significant
clean-up step is necessary after derivatization due to salt
contamination, which often increases sample preparation time,
the opportunity for sample loss, and the analytical variability in
the measurement.
Hydrazone formation is an attractive alternative derivati-
zation strategy in which hydrazide reagents react at the
reducing terminus of glycans much like reductive amination
but does not involve reducing the glycan to a Schiff base
using salts such as sodium borohydride [34]. Thus, when
using hydrazone formation, the product can be directly
injected onto the nano-LC column due to the lack of salts
necessary in the reaction mixture. In addition, 995% reaction
(Gal-GlcNAc) Man GlcNac (NA2) glycan, (NeuAc-Gal-
2 3 2
GlcNAc) Man (Fuc)(GlcNAc) (A2F) glycan, trifluoroace-
2
3
2
tic acid (TFA), acetic acid, ammonium acetate, ethyl
chloroacetate, and β-mercaptoethanol were all purchased
from Sigma Aldrich (St. Louis, MO, USA). Phenylacetic
acid, 3-phenylpropionic acid, 4-phenylbutanoic acid, 5-
phenylpentanoic acid, ethynyl benzene, ethyl 2-(4-bromo-
phenyl)acetate, hydrazine hydrate, 4-phenylpyridine, and ethyl
phenylacetate were purchased from VWR (West Chester, PA,
USA). Peptide: N-Glycosidase F (2.5 mU/μL), denaturing
solution (1 M β-mercaptoethanol and 2% (w/w) SDS), and the
detergent solution (15% Nonidet P40) were purchased from
Prozyme (San Leandro, CA, USA). Pooled human plasma was
purchased from Innovative Research (Novi, MI, USA). High
performance liquid chromatography grade acetonitrile (ACN),
water, and methanol (MeOH) were all purchased from Burdick
and Jackson (Muskegon, MI, USA). IntegraFrit and PicoFrit

S.H. Walker, et. al.: Library of Hydrophobic Hydrazide Reagents
1311
columns were purchased from New Objective (Woburn, MA,
USA) and the TSK-Gel Amide-80 stationary phase was from
TOSOH Bioscience (San Jose, CA, USA). The graphitized
solid phase extraction cartridges were purchased from Alltech
The reaction was then quenched with 500 μL of 0.1% TFA. The
glycans were extracted using graphitized carbon solid phase
extraction (SPE), which has been described in detail previously
[39], and the glycan samples were dried in vacuo.
(
Deerfield, IL, USA).
The derivatization procedure for plasma glycans was
optimized to decrease the amount of peeling for labile capping
sugar units such as sialic acid and fucose. A 1 mg/ml solution
of phenyl2-GPN reagent in 75:25 MeOH:acetic acid solution
was prepared. To the dried glycan sample, 100 μL of the
reagent solution was added and allowed to react for 3 h at 56°C
in an incubator. The glycans were then dried in vacuo and
reconstituted in HILIC initial conditions.
Reagent Synthesis, Purification,
and Characterization
Five hydrazide reagents were synthesized: 2-phenylacetohy-
drazide (GPN), 3-phenylpropanehydrazide (GPN2), 4-phe-
nylbutanehydrazide (GPN3), 5-phenylpentanehydrazide
(
GPN4), and 4-phenethylbenzohydrazide (phenyl2-GPN).
The mono-phenyl reagents were synthesized by Fischer
esterification, converting a carboxylic acid to a methyl ester
followed by reaction with hydrazine hydrate in absolute
ethanol at 90°C to form the hydrazide. The bi-phenyl reagent
Nano-Flow Liquid Chromatography (HILIC)
Separation of derivatized glycans was performed using
online hydrophilic interaction (liquid) chromatography
(HILIC) [39]. An Eksigent nanoLC-2D system equipped
with an AS1 autosampler (Dublin, CA, USA) was used with
a vented-column setup as described previously [40]. TSK-
Gel Amide-80 stationary phase with a 5 μm particle size
(TOSOH Bioscience) was used to pack the trap and
analytical columns, and both were packed in house. The
trap column (360 μm o.d., 100 μm i.d. IntegraFrit) was
packed to ~3.2 cm, and the analytical column (360 μm o.d.,
75 μm i.d. with a 15 μm PicoFrit tip) was packed to ~10 cm.
Mobile phase A and B were 50 mM ammonium acetate (pH 4.5)
and 100% ACN, respectively. Onto the trap, 10 μL of sample
were injected at the initial gradient conditions (20% Solvent A),
washed at high organic solvent (93% Solvent B), and eluted at
500 nL/min. The gradient was ramped from 20% to 60%
Solvent A over 37 min with a total run time of 60 min. A
shallower gradient was used with the plasma glycans that
included an additional washing step in high organic solvent
(93% Solvent B). The gradient for plasma glycans was ramped
from 20% to 55% solvent A over 37 min, and the total run time
was 70 min.
(
phenyl2-GPN) was synthesized by reacting ethynyl benzene
with ethyl 2-(4-bromophenyl)acetate in the presence of a
catalyst and the triple bond was hydrogenated in absolute
ethanol over Pd/C. As with the mono-phenyl reagents the
ester was converted to a hydrazide function via reaction with
hydrazine hydrate in absolute ethanol incubated at 90°C.
Synthesis schemes, purification methods, and characterization
data have been extensively detailed in the supplemental
material.
Reagent Analysis for Model Glycan
A 2 mg/mL solution of each individual reagent was made in
8
5:15 MeOH:acetic acid (vol/vol). The NA2 glycan was
aliquotted into six microcentrifuge tubes (170 pmol in each
tube), dried, and 100 μL (~3500 mol XS) of the synthesized
reagents (GPN, GPN2, GPN3, GPN4, and phenyl2-GPN) were
added to separate dried glycan samples so that five of the tubes
have a different tag in each tube and the final glycan sample has
no tag (only 100 μL of reaction solvent was added). The six
samples were allowed to incubate at 75°C for 3 h, and the
samples were dried in vacuo. A reaction diagram with starting
materials and the final derivatized glycan structure is presented
in the supplemental material (Scheme S1). The samples were
then reconstituted in HILIC initial conditions (80% ACN and
LTQ-FTICR Mass Spectrometry
A hybrid linear ion trap Fourier Transform ion cyclotron
resonance mass spectrometer (Thermo Fisher Scientific, San
Jose, CA, USA) equipped with a 7 Tesla superconducting
magnet was used to acquire precursor and product ion mass
spectra. Electrospray ionization was performed by applying
a 2 kV potential to a zero dead volume union preceding the
trap column. The ions were introduced into a stainless steel
capillary heated to 225°C, and the capillary and tube lens
voltages were set to 42 and 120 V, respectively. The AGC in
20% 50 mM ammonium acetate in water), and the six glycan
samples were combined in an equimolar mixture so that ~1 pmol
of each was injected on column in a 10 μL injection and
analyzed using nanoLC LTQ-FTICR MS.
Cleavage, Derivatization, and Analysis of Plasma
N-linked Glycans
6
the ICR cell was set to 1×10 ions with a maximum
3
Fifty microliters of lyophilized pooled human plasma was
reconstituted in 250 μL of 50 mM tris-HCl buffer (pH 7.5) and
denatured by adding 28 μL of 2% SDS/1 M β-mercaptoethanol
and incubating at 95°C for 5 min. The solution was allowed to
cool to room temperature, and 5 μL of PNGase F (12.5 mU) was
added. The reaction was allowed to proceed for 18 h at 37°C.
injection time of 1 s. In the LTQ, the AGC was set to 8×10
ions with a maximum injection time of 80 ms and a
normalized collision energy of 22. The instrument was
operated in data dependent mode where up to 3 MS/MS
spectra were collected per precursor scan. An include list
was generated consisting of the three most intense m/z

1
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S.H. Walker, et. al.: Library of Hydrophobic Hydrazide Reagents
values in the precursor scan (FTICR), and these ions were
subsequently fragmented (LTQ). If the same m/z value is
chosen twice in 30 s, the m/z was placed on an exclude list
for 30 s. Additionally, a 5000 count threshold was used to
ensure quality MS/MS spectra. The mass spectrometer was
calibrated according to manufacturer protocol and Xcalibur
software (ver. 2.0.5) was used for peak integration and data
analysis.
Results and Discussion
Characterization of Hydrazide Reagents
Five neutral reagents were synthesized in order to increase
the relative abundance of N-linked glycans in ESI MS,
analyze the types of hydrophobic groups that most effec-
tively increase the electrospray response of glycans in MS,
and direct the future synthesis of hydrazide reagents en route
to determining an optimal tag for enhanced glycan analysis
and quantification. Neutral reagents were chosen due to
former studies showing the decreased ion abundance of
glycans derivatized with permanently charged (+) reagents
Non-Polar Surface Area Calculations
The NPSA were calculated as reported previously [5]. Due
to the numerous hydrophilic functions, such as hydroxyl
groups, that comprise glycans, the NPSA of the sugar units
are deemed negligible. Thus, only the NPSA of the hydro-
phobic tagging reagents are taken into account. The NPSA
of the reagents were calculated by using standard Van der
Waal’s radii and bond lengths [41]. Other previous studies
have used computer modeling to integrate the NPSA of
reagents [13] and alternate assumptions [6, 8] in the
determination of NPSA; thus, a detailed description of the
assumptions, formulas, and calculations is included in the
supplemental material (Figure S1) in order to present a
single method for NPSA calculations of hydrophobic
tagging reagents.
[
13]. Furthermore, neutral reagents are compatible in both
the positive and negative ion modes in ESI MS. Since the
negative ion mode has shown to be advantageous to glycan
analysis (more informative fragmentation patterns and the
enhanced analysis of glycans with acidic sugar residues such
as sialic acid) [42–48], the neutral reagents allow one to
explore more experimental space without any additional
sample preparation when switching between positive and
negative modes. Two types of reagents have been synthe-
sized: mono-phenyl and bi-phenyl (Figure 1a). A set of
mono-phenyl reagents was synthesized in order to vary the
alkyl chain length from 1 to 4 methylene units between the
reactive site and the phenyl group. This will allow one to
evaluate the effect of lengthening the hydrocarbon chain
(
a)
(b)
O
H N
O
2
N
H
GPN
GPN2
GPN3
H N
2
N
H
Phenyl-GPNa
O
Aliquot and Tag
Re-Combine
H N
2
N
H
O
+
H N
N
2
N
H
20
18
O
Phenyl-GPa
H N
2
N
H
1
6
O
+
O
14
12
10
8
H N
2
N
N
H
H N
GPa
2
N
H
GPN4
O
CH
3
H N
2
+
N
H
N
CH
3
6
O
CH
3
4
H N
2
N
GTb
H
Phenyl2-GPN
2
0
Previous Reagents
This Study
1
8
20
22
24
26
28
30
32
Retention Time (min)
a
b
Figure 1. (a) The reagents analyzed in this and in previous experiments ( Ref. [13]; Ref. [8]). (b) The EIC’s of each of the
current (boxed) reagents coupled to the NA2 glycan. The HILIC elution order is from the most hydrophobic reagents to the least
(phenyl2-GPN, GPN4, GPN3, GPN2, GPN, native glycan). The relative abundances follow the same trend, as expected

S.H. Walker, et. al.: Library of Hydrophobic Hydrazide Reagents
1313
length with respect to the relative glycan response in ESI-MS.
In addition, another set of molecules has been synthesized that
contains a bi-phenyl reagent. These two types of hydrophobic
groups differ in their types of bonds (σ versus π), geometry
rings to the reagents with the possibility of incorporating
short alkyl chains between the rings.
In order to further characterize the derivatized glycans
and to confirm the stability of the reagents throughout the
sample preparation and MS analysis, the MS/MS spectra of
the two most successful reagents, phenyl2-GPN and GPN4,
are shown in Figure 2a and b, respectively. These spectra
show similar fragmentation in both types of reagents, mono-
and bi-phenyl. In no spectrum has fragmentation of the
reagent been observed, and the tag is never cleaved from the
terminal GlcNAc residue, which allows for the determina-
tion of y and b glycosidic bond cleavages per Domon and
Costello [26]. These fragmentation spectra show that the
composition of unknown glycans can be determined using
either the high mass measurement accuracy of the precursor
scan, by the MS/MS spectra, or a combination of both. The
signal-to-noise (S/N) ratios of the fragmentation spectra show
good quality with only ~1 pmol introduced onto column, and
nearly all the fragmentation products are present. This was not
the case for the native glycans, where several fragmentation
peaks were not able to be distinguished from the noise.
Non-polar surface area (NPSA) calculations are used to
estimate the hydrophobicity of the hydrazide reagents. By
estimating the hydrophobicity of the reagents, the most
successful reagent will be able to be predicted. Figure 3a
demonstrates the correlation between the NPSA calculations
and the experimental ion abundances for the NA2 glycan
derivatized with several different reagents. In general, as the
NPSA, or hydrophobicity, of the reagent is increased, the
signal of the derivatized glycan is increased as well. Based
on this experimental data, future synthetic routes can be
developed based on the NPSA and predict the types of
hydrophobic reagents that will most effectively enhance the
glycan ion abundance. This directed synthetic approach will
allow only the reagents with the best chance of further
increasing glycan ion abundance to be tested, and as the
library continues to grow, the correlation between NPSA and
relative glycan abundance will become more evident and lead
toward the best possible hydrophobic reagent. However,
increasing the hydrophobicity too much will be possible,
leading to analytes that are not retained on a HILIC column or
reagents that are not soluble in the reaction conditions.
(
tetrahedral versus planar), chemical structure, and local-
ization of the electrons. The different characteristics of the
reagents will allow for different solvent-analyte interactions in
the electrospray droplet, and the characteristics most benefi-
cial will be incorporated into future reagents in order to
further enhance the ionization efficiency of glycans.
The synthesis and purification of the reagents were
efficient and were confirmed by the accurate mass spectra of
the derivatized NA2 glycan in the FT-ICR MS. Each reagent
was coupled to the glycan and run individually in order to
determine reaction efficiency and purity (data not shown). All
reagents reacted nearly stoichiometrically with 995% effi-
ciency, and there were no spectra in which there were more
than two N-linked glycan peaks (tagged and free glycan),
implying that there were no impurities or partial synthetic
products present capable of reaction with the glycans. In
addition, this is also evidence that the tags do not degrade in
the reaction conditions or in the ionization source before
analysis in the MS. Several characterization methods have
been employed in order to confirm the synthesis of the
1
13
reagents including H NMR, C NMR, IR, and high
resolution MS, and are included in the supplemental material.
The effects of the hydrophobic hydrazide reagents were
analyzed by creating an equimolar mixture of the NA2
glycan derivatized with each synthesized reagent, and the
derivatized glycans show significant increases in ion
abundance in comparison to the native glycan (Figure 1b).
The extracted ion chromatograms (EIC) of each reagent are
displayed to show the retention time and relative abundance
of each tagged glycan. The reagents synthesized in this
experiment (Figure 1a) were chosen to observe the effective-
ness of incorporating linear alkane hydrophobic groups and
additional phenyl groups onto the reagents. One phenyl
group on the tagging reagent is necessary for the incorpo-
1
3
ration of stable isotopes ( C ) for future quantification
6
studies, but as previously shown, a second phenyl group
significantly increases the ion abundance of tagged glycans
[
13]. It was hypothesized that alkyl groups are more
hydrophobic than phenyl groups due to the delocalization
of the π bonds in the phenyl ring, and a larger increase in
electrospray response would be expected for the reagents
with linear alkanes. As the number of methylene groups is
increased, the ion abundance increased, although it was not
until the addition of three additional methylene groups
Separation by HILIC allows for another metric for the
estimation of the hydrophobicity of the species present. In
HILIC, the more hydrophobic species elute earlier, allowing
for an estimation of the hydrophobicity of the analytes based
on the retention time. Though HILIC is considered to be a
“mixed-mode” separation method where both partitioning
and adsorption mechanisms are involved in separation [49–
52], the reagents used in this experiment are chemically
similar and, thus, the retention mechanisms are assumed to
be similar. Figure 3b is a plot of retention time (RT) versus
NPSA and shows an additional correlation between the
experimental (RT) and calculated (NPSA) hydrophobicity.
The two types of reagents, mono- and bi-phenyl, both show
linear trends in which the retention time increases as the
(
GPN4) that the electrospray response of the derivatized
NA2 glycan increased equivalently to that of the reagent
with two phenyl groups and no methylene groups (phenyl-
GPN). In addition, the combination of two additional alkyl
groups and two phenyl groups produced a reagent that
outperformed any reagent synthesized to date. This evidence
leads one to infer that the best method to increase the
electrospray response of glycans is to add several phenyl

1
314
S.H. Walker, et. al.: Library of Hydrophobic Hydrazide Reagents
H
N
(a)
+ 2+
electrons, and the geometry of the reagents are the differ-
ences between the two analytes and interact in different
ways with the stationary phase. Though HILIC separation
mechanism, retention times, and stationary phase interac-
[
M+2NH4
]
N
H
O
+
+ 2+
[
M+H +NH
]
4
[
M+2H+]2+
9
35 940 945 950 955 960 965
(a)4.5
m/z
NEUTRAL
4
Phenyl2-GPN
H
N
O
N
H
3
2
.5
3
H
N
GPN4
O
N
H
Phenyl-GPN
GPN
.5
2
GPN3
GPN2
a
4
00
600
800
1000 1200 1400
Phenyl-GP
m/z
1.5
1
H
N
CHARGED
[
M+2NH +]2+
N
H
(b)
4
b
a
O
0.5
0
GP
GT
+
+ 2+
[
M+H +NH4 ]
[
M+2H+]2+
(
b) 28
H
N
N
H
O
905 910 915 920 925 930
m/z
GPN
2
2
2
2
1
6
4
2
0
8
GPN2
GPN3
NEUTRAL
H
N
N
H
O
GPN4
Phenyl-GPN
Phenyl2-GPN
Phenyl4-GPN
4
00
600
800
1000
1200
1400
m/z
c
Figure 2. The MS/MS data of the (a) phenyl2-GPN and (b)
GPN4 reagents. The two fragmentation spectra show similar
patterns, and in no case was a loss or fragmentation of the
tagging reagent observed. In addition, the composition of the
glycan was able to be determined using the MS/MS spectra.
The insets are the high mass measurement accuracy FT-ICR
precursor ions, showing the monoisotopic peak ([M + 2H ]
and the ammonium adduction peaks ([M + H + NH
M + 2NH ] ). In both (a) and (b), squares and circles denote
N-acetyllactosamine (HexNAc) and hexose (Hex) monosac-
charide residues, respectively
7
5
100
125
150
175
200
225
Non-Polar Surface Area (Å2)
+
2+
)
Figure 3. (a) The abundance of the derivatized glycan
relative to the free glycan) has been plotted versus the
NPSA of the reagents. Diamonds denote neutral reagents,
and squares represent charged reagents ( Ref. [8]; Ref. [13]).
b) The retention time of the derivatized glycan is plotted
+
+ 2+
4
]
and
(
+
4
2+
[
a
b
(
versus the NPSA of the reagents. Diamonds represent mono-
phenyl reagents, and triangles represent bi-phenyl reagents.
The error bars in both (a) and (b) indicate 95% confidence
reagents become less hydrophobic. However, the slopes and
intercepts of these lines differ, which implies slightly
different separation mechanisms between the two types of
reagents coupled to glycans. Though the reagents GPN4 and
phenyl-GPN have nearly identical NPSA, the retention time
between the two derivatized glycans is significantly differ-
ent. Thus, the different types of bonds, the localization of
c
intervals (3 ≤ n ≤ 5). The phenyl4-GPN reagent was
synthesized (identical to phenyl2-GPN, except that it con-
tains a 4-C chain between the phenyl rings), but the
recrystallization purification was only minimally successful,
and thus, it was analyzed for retention time purposes only
and not for quantitative analysis

S.H. Walker, et. al.: Library of Hydrophobic Hydrazide Reagents
1315
100
H
N
N
O
H
7
5
A2F Glycan + Tag
> 82%
H
N
N
H
O
O
-
or -
5
0
5
0
H
N
N
H
“
Peeling” of Sialic Acid
or Fucose Residues
A2F Glycan
2
<
3%
<
15%
35
40
45
50
55
Retention Time (min)
Figure 4. The EIC of the native and tagged A2F glycan after derivatization with phenyl2-GPN at 56°C for 3 h in 25:75 acetic
acid:MeOH. This shows 995% conversion to the derivatized glycan, however there is still a small amount of peeling, or loss of
sialic acid or fucose
tions are not fully understood, this study shows that with all
other properties held constant, the glycans derivatized with
reagents primarily composed of alkyl chains interact more
with the stationary phase than glycans derivatized with the
bi-phenyl reagents.
were cleaved, derivatized with phenyl2-GPN, and analyzed.
Initially, intact sialylated glycans were not able to be
detected. Upon further investigation, it was determined that
the high reaction temperature combined with acidic con-
ditions (75°C in 15% acetic acid for 3 h) was causing
peeling, or loss of labile sugar residues (most notably
NeuAc). Thus, numerous reaction conditions were tested using
a model glycan, A2F, which contains two terminal sialic acid
residues and one core fucose. It was found that a lower
temperature and higher acidity (56°C in 25:75 acetic acid:
MeOH for 3 h) provided near complete conversion to the
derivatized glycan (995%) and minimized the peeling (Figure 4).
Analysis of the Human Plasma N-Linked Glycome
Using the Phenyl2-GPN Reagent
In order to demonstrate the compatibility and effectiveness
of the novel glycan hydrophobic reagents in complex
mixtures, the N-linked glycans from pooled human plasma
(
a)
Phenyl-2GPN derivatized N-linked
(b)
glycan elution window
H
N
1
00
N
H
O
(
magnified x 10)
7
5
25
30
35
40
45
50
55
60
Retention Time (min)
H
N
5
2
0
5
N
H
O
Native N-linked glycan
(c)
elution window
[M+H
+
+NH4
+ 2+
]
[
M+2NH + ]2+
4
(magnified x 10)
[
M+2H+]2+
[
M+H +NH4 ]2+
+
+
[
M+2H+]2+
0
2
0
22
24
26
28
30
32
34
36
38
1560
1570
m/z
1580 1440
1450
m/z
1460
Retention Time (min)
Figure 5. The compatibility of the phenyl2-GPN hydrophobic hydrazide reagent with glycans cleaved from complex mixtures:
a) the base peak chromatogram of the derivatized N-linked glycans (solid line) overlaid with the base peak chromatogram of
(
the native N-linked glycans (dotted line); an average of 4-fold increase in glycan ion abundance is observed for all glycan
species. (b) the EIC and reaction efficiency of the NA2 glycan cleaved from plasma proteins; (c) the mass spectra of the tri-
sialylated A3 glycan both native and derivatized

1
316
S.H. Walker, et. al.: Library of Hydrophobic Hydrazide Reagents
These conditions were used in all the subsequent studies
containing complex glycans with sialic acid or fucose residues.
Figure 5 shows the analysis of N-linked glycans cleaved
from plasma, both native and derivatized. The base peak
chromatograms have been overlaid in Figure 5a and the
derivatized glycans show on average a 4-fold increase in total
ion abundance with certain negatively charged glycans show-
ing up to a 10-fold increase. Figure 5b shows the derivatization
efficiency of the glycans in a complex mixture. The 997%
reaction efficiency in plasma displays the robustness of the
derivatization even in the most complex of samples, plasma.
Additionally, Figure 5c demonstrates the advantage of deriva-
tizing negatively charged sialylated glycans, which are often
difficult to ionize in the positive ion mode. When analyzing
native glycans, the tri-sialylated A3 glycan is only minutely
detected. It is shown that the signal of the native A3 glycan is
only slightly above the noise, whereas 910-fold S/N is detected
for the derivatized A3 glycan. This provides more statistically
accurate isotopic ratios, which yield higher quality data and
will lead to the accurate quantification of N-linked glycans over
a wider dynamic range.
Biotechnology Training Program (S.H.W.), the W. M. Keck
Foundation, and North Carolina State University.
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The hydrazide reagents are effective in increasing the glycan
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