Facile synthesis of alumina hollow spheres for on-plate-selective enrichment of phosphopeptides

Article abstract of DOI:10.1039/c0cc05524g

Alumina hollow spheres were synthesized, spotted and sintered on a stainless-steel plate. These spots were used to selectively capture phosphopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI-MS. This provides a fast and efficient on-plate selective enrichment method for phosphopeptides investigation.

Full text of DOI:10.1039/c0cc05524g

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Facile synthesis of alumina hollow spheres for on-plate-selective
enrichment of phosphopeptidesw
Jin Lu, Shasha Liu and Chunhui Deng*
Received 13th December 2010, Accepted 15th March 2011
DOI: 10.1039/c0cc05524g
Alumina hollow spheres were synthesized, spotted and sintered
on a stainless-steel plate. These spots were used to selectively
capture phosphopeptides from peptide mixtures and the captured
target peptides could be analyzed by MALDI-MS. This provides
a fast and efficient on-plate selective enrichment method for
phosphopeptides investigation.
of phosphopeptides,5 while Tang et al. functionalized the
MALDI target plate with boronic acid-modified gold nano-
particles for selective enrichment of glycopeptides.6 However,
to our knowledge, there is no on-plate strategy using hollow
spheres proposed for pre-treatment of phosphopeptides in
biological samples. Due to the special structure of hollow
spheres, a higher surface area can be obtained which makes
it highly efficient for desired species. So these hollow spheres
have been widely studied recently and have the potential for
diverse applications, such as in gas sensitivity or catalysis,
or as advanced ceramic materials.7 Besides, the on-plate
enrichment strategy makes the analysis more speedy and
high-throughput. Thus it can be seen that an on-plate
enrichment strategy using hollow spheres can be a promising
way for highly selective enrichment of phosphopeptides.
Herein, a new plate-based technique for fast enrichment of
phosphopeptides is described. The MALDI target plate is
covered with our new synthesized alumina hollow spheres
and analyzed by MALDI-TOF-MS after enriching phospho-
peptides from peptide mixtures. Owing to the special hollow
structure, a higher surface area was obtained which makes it
highly efficient for desired phosphopeptides. As a result, this
enrichment approach has proven to be effective and sensitive
for analysis of phosphopeptides from peptide mixtures and
provides a promising way to establish an online investigation
strategy for phosphopeptides of biological samples.
Protein phosphorylation, the most important and ubiquitous
post-translational modification of proteins, is able to regulate
almost all aspects of cell life in both prokaryotes and eukaryotes.
With the motivation of understanding these bioprocesses,
much effort has been devoted to the development of methods
to identify the specific sites of phosphorylation.1 Recently, the
mass spectrometric technique has emerged as a powerful tool
for phosphorylation site mapping.2 However, the signals of the
phosphopeptides are often suppressed by nonphosphorylated
peptide residues contained in protein digests, which means
that the identification of phosphorylated peptides is still
challenging. Therefore, separation and enrichment of
phosphorylated peptides from proteolytic digest mixtures is
in urgent demand. To meet this requirement, a variety
of separation techniques have been developed, including
immunoprecipitation, chemical-modification strategies, strong
cation exchange chromatography, immobilized metal ion
affinity chromatography (IMAC) and metal oxide affinity
chromatography (MOAC).3 Among these techniques, IMAC
and MOAC are the most simple and convenient and have been
widely studied. Recently, on-plate selective enrichment has
attracted much attention because of the fact that immobilization
of either IMAC materials or metal oxides on matrix-assisted
laser desorption/ionization (MALDI) plates allows for on-probe
purification that can lead to high-throughput analyses and
reduce sample loss due to adsorption on container walls. For
example, Tan et al. modified the MALDI target plate
with magnetic affinity nanoparticles for specific capture of
phosphopeptides.4 Qiao et al. functionalized a MALDI
target plate by sintering TiO2 nanoparticles for enrichment
The synthesis strategy of alumina hollow spheres is
shown in Scheme 1. First, carboxylate-rich carbonaceous
microspheres were synthesized according to a reported work.8
Briefly, glucose is used as the carbon source and its concentra-
tion in water is 10 w/v%, and acrylic acid is added to make the
concentration of acrylic acid 10 w/v%. The reaction mixtures
were then transferred into
a Teflon-lined stainless-steel
autoclave with a capacity of 200 mL. The autoclave was sealed
and heated at 190 1C for 16 h and naturally cooled to room
Department of Chemistry & Institutes of Biomedical Science,
Fudan University, Shanghai 200433, China.
E-mail: chdeng@fudan.edu.cn; Fax: +86-21-65641740
w Electronic supplementary information (ESI) available: Experimental
details, XRD pattern, FTIR spectra, MS. See DOI: 10.1039/
c0cc05524g
Scheme 1 The synthetic strategy of alumina hollow spheres.
c
5334 Chem. Commun., 2011, 47, 5334–5336
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temperature. Then the obtained materials were washed several
times with water and dried under vacuum at 50 1C for 12 h for
further use. Carboxylate-rich carbonaceous microspheres
(0.1 g) were ultrasonicated in a 40 mL 0.2 M Al(NO3)3
solution for 70 min, followed by washing with deionised water
three times. The product was then dried under vacuum at
50 1C and calcined in nitrogen at 500 1C for 1 h. After the
furnace was left to cool to room temperature, the final
products were transferred to tubes and preserved under room
temperature.
The morphology of as-prepared carboxylate-rich carbo-
naceous microspheres and alumina hollow spheres were
obtained by a scanning electron microscope (SEM) and
transmission electron microscope (TEM). As shown in
Fig. 1a and c, both of the carboxylate-rich carbonaceous
microspheres and alumina hollow spheres are monodispersed
and have a nearly uniform size. From Fig. 1b and d, the TEM
observations indicate that the diameter of the obtained
carboxylate-rich carbonaceous microspheres is about 2 mm
and that of alumina hollow spheres is about 700 nm. The size
of the hollow spheres was about 40% of that of the original
templates, confirming previous results.9 Fourier transform
infrared (FT-IR) spectroscopy (Fig. S1, ESIw) was employed
to further characterize the as-synthesized carboxylate-rich
carbonaceous microspheres. The absorption bands at B1700
indicate the presence of a large amount of carboxyl groups.
The other absorption bands belong to CQC double bonds
(1618 cmÀ1), C–OH stretching and OH bending vibrations
(1000–1300 cmÀ1) and the adsorption bands at B3400 cmÀ1
belong to the residual hydroxyl groups constituting the hydro-
philic surface. The wide-angle X-ray diffraction (WAXRD)
pattern (Fig. S2, ESIw) indicates that the alumina hollow
spheres are amorphous. Fig. 2 displays the N2 adsorption–
desorption isotherm and the corresponding pore size distribution
curve for the alumina hollow spheres. The BET surface area
and total pore volume were calculated to be 228.9 m2 g–1 and
1.17 cm3 g–1, respectively, indicating that the sample has a
relatively high surface-to-volume ratio.
Fig. 2 N2 adsorption–desorption isotherms and pore size distribution
(insert) of the alumina hollow spheres.
To modify the target plate, drops of a suspension of the
alumina hollow spheres were spotted on the plate, followed
by heating at 200 1C for sintering, which is an important
procedure to form a stable frit support phase for samples and
matrix. This on-plate enrichment strategy was studied by
extracting phosphopeptides from tryptic digests of b-casein,
which is commonly contaminated with a trace of a-casein. A
direct analysis of 40 fmol b-casein by MALDI-TOF MS
indicates that nonphosphopeptides dominate the spectrum
and nearly no phosphopeptides were detected (Fig. 3a).
However, after enrichment by alumina hollow spheres, the signals
Fig. 3 MALDI mass spectrum of peptides derived from b-casein: (a)
without enrichment (b) enriched by alumina hollow spheres. Phos-
phopeptide ions identified are marked with numbers. * Phosphopeptides
of b-casein, ^ phosphopeptides of a-casein, # the metastable losses of
phosphoric acid.
Fig. 1 (a) SEM image and (b) TEM image of carboxylate-rich
carbonaceous microspheres. (c) SEM image and (d) TEM image of
alumina hollow spheres.
c
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Besides, we used another protein sample ovalbumin to
further confirm the usefulness of this method and successfully
enriched one phosphopeptide. The detailed information is
described in the ESI.w The results suggest that the on-plate
enrichment strategy using alumina hollow spheres we reported
here is a promising way for enrichment of phosphopeptides.
In summary, a novel on-plate selective enrichment approach
is proposed. A MALDI target plate was sintered with alumina
hollow spheres to form a thin layer, and then the plate was
utilized to specifically concentrate and selectively enrich
phosphopeptides from complex peptide mixtures without
additional procedures. Captured phosphopeptides on the plate
can be submitted directly for MALDI-TOF MS analysis after
deposition of 2,5-dihydroxybenzoic acid as the MALDI
matrix. Finally, we have demonstrated that this alumina
hollow spheres modified target plate exhibits high sensitivity
and selectivity to phosphopeptides. Besides, the on-plate
enrichment and detection steps are significantly simple and
high-throughput. Moreover, the sample amount needed
during the analytical procedure is extremely small. All of these
strong points make this on-plate enrichment strategy a
promising way for a high-throughput and automatic online
analysis of phosphopeptides.
Fig. 4 MALDI mass spectrum of peptides derived from a peptide
mixture of b-casein and BSA at a molar ratio of 1 : 10: (a) without
enrichment (b) enriched by alumina hollow spheres. * Phosphopeptides
of b-casein, # the metastable losses of phosphoric acid.
for the b-casein phosphopeptides (marked with an asterisk)
significantly increase and dominate the spectrum (Fig. 3b).
Furthermore, three phosphopeptides derived from a-casein
(marked with a circumflex) were also observed and nearly
no nonphosphorylated peptides were observed, indicating
high enrichment efficiency of the alumina hollow spheres.
The signals marked with a hash could be assigned to a
dephosphorylated fragment of the phosphopeptide through
loss of H3PO4. The observed phosphopeptides are marked
with numbers and the detailed information of the observed
phosphopeptides is listed in Table S1, ESIw. To confirm that
the large surface area can benefit the enrichment efficiency,
we did a comparison experiment using mesoporous TiO2
microspheres we synthesized before, whose specific surface
This work was supported by the National Natural
Science Foundation of China (Project No. 20875017), the
Technological
Innovation
Program
of
Shanghai
(09JC1401100), the National Basic Research Priorities
Program (Project No. 2007CB914100/3), and Shanghai
Leading Academic Discipline Project (B109).
Notes and references
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area was 84.98 m2 gÀ1 10 There were several nonphosphorylated
.
peptides in the MALDI mass spectrum of peptides derived
from b-casein after enriching by TiO2 microspheres and the
signal/noise is much lower than that by alumina hollow
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The selectivity of this on-plate enrichment method was
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differences suggest that alumina hollow spheres could
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To study the detection limit of the alumina hollow spheres
used for enrichment of phosphopeptides, we applied the
alumina hollow spheres to the enrichment from b-casein digest
with different concentrations. When the concentration of the
b-casein digest is 5 fmol, the ion signals from the phosphopeptides
can still be detected, which indicates the high detection
sensitivity of alumina hollow spheres (see Fig. S4, ESIw).
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c
5336 Chem. Commun., 2011, 47, 5334–5336
This journal is The Royal Society of Chemistry 2011