Highly specific enrichment of N-glycoproteome through a nonreductive amination reaction using Fe3O4@SiO2-aniline nanoparticles

Article abstract of DOI:10.1039/c4cc10285a

A novel method based on the conjunction of aldehydes from oxidized glycopeptides to aniline groups on magnetic nanoparticles via nonreductive amination is reported for the highly selective enrichment of N-glycopeptides. For the first time, a nonreductive amination reaction has been introduced into N-glycoproteome extraction, and correspondingly a new type of aniline-functionalized nanoparticle has been designed and synthesized.

Full text of DOI:10.1039/c4cc10285a

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Highly specific enrichment of N-glycoproteome
through a nonreductive amination reaction using
Fe3O4@SiO2-aniline nanoparticles†
Cite this:DOI: 10.1039/c4cc10285a
Received 24th December 2014,
Accepted 13th February 2015
Ying Zhang,‡ab Meng Yu,‡c Cheng Zhang,ab Yali Wang,a Yi Di,b Changchun Wang*c
and Haojie Lu*ab
DOI: 10.1039/c4cc10285a
www.rsc.org/chemcomm
A novel method based on the conjunction of aldehydes from enrichment strategy.6 However, this method requires a long
oxidized glycopeptides to aniline groups on magnetic nanoparticles conjugation time (12–16 h) to immobilize the N-glycopeptides
via nonreductive amination is reported for the highly selective onto the solid phase, although a recent study showed the addition
enrichment of N-glycopeptides. For the first time, a nonreductive of aniline can accelerate the coupling time.7 Meanwhile, the
amination reaction has been introduced into N-glycoproteome harsh washing process required to remove nonspecific adsorp-
extraction, and correspondingly a new type of aniline-functionalized tions inevitably gives rise to a loss of sample, limiting the
nanoparticle has been designed and synthesized.
sensitivity of this method. Recently, we have developed a new
N-glycoproteome enrichment method based on the conjunction
N-Glycosylation is one of the most common protein post- of aldehydes from oxidized N-glycopeptides to an amine group on
translational modifications.1 Changes in N-glycoprotein are the magnetic nanoparticles via a reductive amination reaction,
associated with various disease progression, and many clinical which significantly shortens the conjugation time to less than
biomarkers are N-glycoproteins.2 Therefore, the highly sensitive 4 h.8 However, the requirement for a reductive reagent to generate
identification of N-glycoproteome has the potential for use in the stale bond between the N-glycopeptides and the amine-
disease diagnosis, prognosis, and the prediction of treatments, functionalized magnetic nanoparticles represents a major limitation
but there is still many challenges before it can be realized.3 The of this method. To overcome this disadvantage, we have begun to
human proteome database contains thousands of predicted investigate alternative methods.9 Compared with the aliphatic Schiff
N-glycosylation sites; however, only 5% of the N-glycosylation base, the aromatic Schiff base offers better stability, because of its
sites have been experimentally proven so far, mainly due to the conjugation structure, and it can remain as a stable Schiff base in a
inherently low availability of N-glycoproteome.4 The selective wide pH range without reducing the CQN bond to C–N bond.10
enrichment of N-glycopeptides has become a prerequisite for Also, it is documented that the aniline can react with the aldehyde
mass spectrometry (MS)-based N-glycoproteome studies, because group under nonreductive amination conditions without the
fishing out the N-glycoproteome before the MS analysis can addition of a reductive reagent.11 The omission of the reducing
make the characterization of N-glycoproteins more specific.5
reagent would significant simply the whole process and improve
To meet the pressing need in MS-based N-glycoproteome the enrichment sensitivity. To the best of our knowledge, this is
research, numerous N-glycopeptides enrichment methods have the first time that the nonreductive amination reaction has been
been developed. Among these, hydrazide-chemistry-based solid introduced into N-glycoproteome enrichment.
phase extraction, which relies on the conjugation of the glycans
to a hydrazide-functionalized solid support, is the most specific functionalized nanomaterial that can capture N-glycoproteome
through nonreductive amination reaction was precisely
To develop such a new enrichment strategy, a novel aniline-
a
designed and synthesized. The design requires that first, the
aniline-functionalized nanoparticles should be easily prepared
with high efficiency. Second, such composite particles should
display a strong response to an external magnetic field, because
this feature would enable us to capture and release the particles
easily. The synthesis of Fe3O4@SiO2-aniline is shown in Scheme 1.
A silane coupling reagent with a nitrobenzol group was synthesized
through modifying the (3-aminopropyl)triethoxysilane with p-nitro-
benzoic acid and was then chromatographically purified, and
a Shanghai Cancer Center and Department of Chemistry, Shanghai 200032,
P. R. China. E-mail: luhaojie@fudan.edu.cn
b Institutes of Biomedical Sciences and Key Laboratory of Glycoconjuates MOH,
Fudan University, Fudan University, Shanghai, 200032, P. R. China
c State Key Laboratory of Molecular Engineering of Polymers and Department of
Macromolecular Science, Laboratory of Advanced Materials, Fudan University,
Shanghai 200433, P. R. China. E-mail: ccwang@fudan.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc10285a
‡ Y. Z. and Y. M. contributed equally.
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For the Fe3O4@SiO2 nanoparticles, the strong IR band at 567 cmÀ1
is attributed to the vibration of the Fe–O, and the adsorption peak
at 1087 cmÀ1 is attributed to the Si–O–Si vibration, indicating
the successful preparation of the Fe3O4@SiO2 nanoparticles. Com-
paring the FT-IR spectrum of the Fe3O4@SiO2-aniline with the
Fe3O4@SiO2, new absorption peaks at 1600 cmÀ1 and 1520 cmÀ1
,
which are attributed to the vibrations of the aromatic ring and the
bending vibration of N–H, appeared in the FT-IR spectrum of
Fe3O4@SiO2-aniline, indicating the successful modification of
NBTES. The ZETA potential was measured to further verify the
reduction of nitrobenzol group into aniline group (Table S1, ESI†).
The ZETA potential of Fe3O4@SiO2-nitrobenzol was À16.3 mV.
After the reduction by zinc powder, due to the positive elec-
tricity of aniline, the ZETA potential of Fe3O4@SiO2-aniline
increased to +6.3 mV, indicating the successful reduction.
The transmission electron microscopy (TEM) images (Fig. 1c
and d) reveal the iron oxide microspheres are well encapsulated
in a condensed, amorphous silica shell. Additionally, the super-
paramagnetic core (with a VSM of 35 emu gÀ1, Fig. 1b) makes the
Fe3O4@SiO2-aniline material able to be readily separated from the
solution phase within 30 s with the help of a magnet and then
quickly redispersed after removal of the magnetic field. This
excellent magnetic response and redispersibility of Fe3O4@SiO2-
aniline material would contribute to an efficient enrichment and
separation of the N-glycoproteome.
The feasibility of introducing the nonreductive amination
reaction into N-glycoprotein enrichment was demonstrated first by
analyzing a model N-glycoproteins asialofetuin (ASF) with three
known N-glycosylation sites. In a proof-of-concept experiment, the
enrichment procedure was determined, and is illustrated in
Scheme 2, and the proposed main reaction pathways of the
nonreductive amination reaction are shown in Fig. S2 (ESI†). The
comparison of this new method with the reductive amination
reaction-based method and the traditional hydrazide chemistry-
based enrichment methods is shown in Fig. S3, ESI.† In a typical
enrichment procedure, the peptide digests of ASF were first oxi-
dized by NaIO4, then dissolved in a loading buffer and incubated
with the Fe3O4@SiO2-aniline to couple the N-glycopeptides onto the
Fe3O4@SiO2-aniline nanoparticles through a nonreductive amina-
tion reaction. Afterward, the Fe3O4@SiO2-aniline with the captured
N-glycopeptides was separated from the mixed solution using an
external magnetic field, and then the nanoparticles were washed
with different types of buffers several times to remove the non-
specifically adsorbed peptides and other impurities. Finally, the
glycopeptides were released from the Fe3O4@SiO2-aniline with
PNGase F for MS analysis. First, we focused on investigating the
coupling condition, including the reaction buffer, the reaction
temperature and the reaction time, to efficiently conjugate the
oxidized N-glycopeptides onto the Fe3O4@SiO2-aniline nano-
particles. We found that the conjugation reaction proceeded
well in 70% methanol and 30% acetic acid (v/v) at 60 1C for 4 h,
with all of the six theoretical deglycosylated glycopeptides peaks
detected (Fig. S4–S6, ESI†). The optimized mass ratio of the solid
phase to sample was determined as 1 mg of Fe3O4@SiO2-aniline
per 1 mg of protein digests (Fig. S7, ESI†). Then, we compared the
effect of the reducing reagent on the enrichment performance,
Scheme 1 Synthesis scheme for Fe3O4@SiO2-aniline.
herein, denoted as NBTES. The Fe3O4@SiO2 core–shell nanoparticle
for modifying the NBTES silane coupling reagent was synthesized
through a modified solvothermal reaction, as in previous work, and
then the silica shell was coated on the surface of the Fe3O4 cores.8
Subsequently, the NBTES was used to modify the as-prepared
Fe3O4@SiO2 nanoparticles through a one-step silylation reaction to
obtain Fe3O4@SiO2-nitrobenzol. The final product of Fe3O4@SiO2-
aniline was obtained by treating the Fe3O4@SiO2-nitrobenzol with
zinc powder in ammonium chloride aqueous solution to reduce the
nitrobenzol groups to aniline groups.
The successful synthesis of these Fe3O4@SiO2-aniline nano-
particles was proven by various means. The NMR spectra (Fig. S1,
ESI†) clearly demonstrate the successful synthesis of the nitrobenzol
silane coupling agents, and the detailed peaks attribution is shown
in the ESI.† To prove the functionalization of the aniline group on
the nanoparticles, Fourier transform infrared (FT-IR) spectroscopy
was used to characterize the NBTES, Fe3O4@SiO2 nanoparticles, and
the Fe3O4@SiO2-aniline nanoparticles, respectively, see Fig. 1(a).
NBTES was characterized first; the peaks at 1640 cmÀ1 and
1520 cmÀ1 corresponding to the stretch vibration of CQO and
the bending vibration of N–H proved the structure of NBTES.
Fig. 1 (a) FTIR spectroscopy of (i) NBTES, (ii) Fe3O4@SiO2 and (iii) Fe3O4@-
SiO2-aniline; (b) magnetic hysteresis curves of (i) Fe3O4, (ii) Fe3O4@SiO2
and (iii) Fe3O4@SiO2-aniline; TEM images of (c) Fe3O4 and (d) Fe3O4@SiO2
the scale bar is 100 nm.
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Fig. 2 MALDI-TOF mass spectra of a tryptic digest of ASF (a) before
isolation but following deglycosylation by PNGase F and (b) after isolation
by Fe3O4@SiO2-aniline and then followed by deglycosylation by PNGase F.
The symbols ‘‘#’’ represents the deglycosylated glycopeptides (including
fragments from deglycosylated glycopeptides).
Scheme 2 The enrichment and MS analysis scheme for N-glycopeptides
using Fe3O4@SiO2-aniline.
enrichment, the signals of N-glycopeptides were hardly seen,
due to the interference by significant amounts of nonglyco-
and found that the omission of the reducing reagent NaBH3CN peptides from MYO (Fig. S9a, ESI†). After enrichment, the
did not hamper the enrichment performance (Fig. S8, ESI†). signals of glycopeptides were easily detected with a clean
The reason for is that, different from the reaction between the background (Fig. S9b, ESI†). As the mass spectrum revealed,
aliphatic amine and aldehyde, the condensation product between the glycopeptides were selectively fished out from the mixture
the aniline and aldehyde can form as a stable Schiff base without by the Fe3O4@SiO2-aniline. This result indicated the excellent
reducing the CQN bond to C–N bond. On the contrary, the enrichment selectivity of Fe3O4@SiO2-aniline toward N-glycopeptides.
addition of a reducing reagent requires an additional desalting The selectivity is comparable to our previous developed method.9 This
process before the MS analysis.
new method also possesses high sensitivity at the nanogram per
The established new enrichment method shows remarkable microliter level. To investigate the sensitivity of the established
selectivity toward N-glycoproteins, as detailed in the following. method, a series of diluted oxidized asialofetuin digest solutions were
After optimization of the enrichment procedure, the ASF tryptic tested and the lowest detectable concentration was estimated to be at
digests were subjected to the enrichment process, and ASF 5.0 ng mLÀ1 (Fig. S10, ESI†). This sensitivity is 2–5 times better than
tryptic digests without enrichment were analyzed directly our previously developed methods. The above results make us believe
for comparison. The standard N-glycoprotein ASF harbors three that this novel protocol can extract N-glycoproteome with remarkable
N-glycosylation sites at N99CS, N176GS, and N156DS; however, specificity and high sensitivity.
due to the presence of nonglycopeptides, only four deglycosy-
The utility of this nonreductive amination reaction-based
lated N-glycopeptides at m/z: 1627.81, 1754.79, 1780.80, and enrichment method is further demonstrated by profiling the
3017.54 (including only two of the N-glycosylation sites) N-glycoproteome of a normal human serum sample. The
appeared in the spectrum after the deglycosylation by PNGase serum sample from a normal volunteer was provided by Fudan
F, while peaks of the nonglycopeptides clearly dominated University Shanghai cancer center. The research followed the
the spectrum (Fig. 2a). After enrichment and deglycosylation tenets of the Declaration of Helsinki and was approved by the
by PNGase F, six predominant peaks of the deglycosylated Ethics Committee of Fudan University Shanghai cancer center.
N-glycopeptides from ASF were detected with a very clean After reduction and alkylation, the serum sample was digested
background, covering all of the three known glycosylation sites into peptides, followed by oxidation, enrichment by Fe3O4@
(Fig. 2b). The enrichment result clearly confirmed the selectivity SiO2-aniline, deglycosylation and then 1D nano-LC-MS/MS
of this method. To further investigate the selectivity of this analysis. In total, 156 unique N-glycosylation sites were found
method, a mixture of the tryptic digests of a standard nonglyco- in 148 glycopeptides, which were assigned to 80 glycoproteins.
protein myoglobin (MYO) and ASF was prepared at a molar The specificity of the identified glycoproteins was as high as
ratio of MYO : ASF at 100 : 1 to mimic a real sample. Before 71.8%. The detailed information is provided in Table S2 (ESI†).
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It should be noted that only 1 mL of sample was used in our 21375026), and Shanghai Projects (Grants Eastern Scholar,
experiments, which is a very small sample volume. The results 11XD1400800, 13520720200, Shanghai Rising-Star and B109).
of a real sample analysis clearly indicated the selectivity and
sensitivity of this new method.
Notes and references
In summary, a new aniline-functionalized nanoparticle
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This work was supported by the National Science and Technology
Key Project of China (2012CB910602 and 2012AA020204), the National
Science Foundation of China (Grant No. 21335002, 51073040, and
Chem. Commun.
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