A Two-Dimensional Metallacycle Cross-Linked Switchable Polymer for Fast and Highly Efficient Phosphorylated Peptide Enrichment

Article abstract of DOI:10.1021/jacs.0c12904

The selective and efficient capture of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis. Here we report a new switchable two-dimensional (2D) supramolecular polymer that serves as an ideal platform for the enrichment of phosphopeptides. A well-defined, positively charged metallacycle incorporated into the polymer endows the resultant polymer with a high affinity for phosphopeptides. Importantly, the stimuli-responsive nature of the polymer facilitates switchable binding affinity of phosphopeptides, thus resulting in an excellent performance in phosphopeptide enrichment and separation from model proteins. The polymer has a high enrichment capacity (165 mg/g) and detection sensitivity (2 fmol), high enrichment recovery (88%), excellent specificity, and rapid enrichment and separation properties. Additionally, we have demonstrated the capture of phosphopeptides from the tryptic digest of real biosamples, thus illustrating the potential of this polymeric material in phosphoproteomic studies.

Full text of DOI:10.1021/jacs.0c12904

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A Two-Dimensional Metallacycle Cross-Linked Switchable Polymer
for Fast and Highly Efficient Phosphorylated Peptide Enrichment
Li-Jun Chen, Sean J. Humphrey, Jun-Long Zhu, Fan-Fan Zhu, Xu-Qing Wang, Xiang Wang, Jin Wen,*
Hai-Bo Yang,* and Philip A. Gale*
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ABSTRACT: The selective and efficient capture of phosphopeptides is
critical for comprehensive and in-depth phosphoproteome analysis. Here we
report a new switchable two-dimensional (2D) supramolecular polymer that
serves as an ideal platform for the enrichment of phosphopeptides. A well-
defined, positively charged metallacycle incorporated into the polymer endows
the resultant polymer with a high affinity for phosphopeptides. Importantly,
the stimuli-responsive nature of the polymer facilitates switchable binding
affinity of phosphopeptides, thus resulting in an excellent performance in
phosphopeptide enrichment and separation from model proteins. The polymer
has a high enrichment capacity (165 mg/g) and detection sensitivity (2 fmol),
high enrichment recovery (88%), excellent specificity, and rapid enrichment
and separation properties. Additionally, we have demonstrated the capture of
phosphopeptides from the tryptic digest of real biosamples, thus illustrating
the potential of this polymeric material in phosphoproteomic studies.
through coordination-driven self-assembly.20−25 These systems
have found applications in molecular recognition, sensing,
catalysis, biomedicines, and other areas.26−30 More recently,
significant progress has been made on the functionalization of
coordination metal complexes to construct stimuli-responsive
supramolecular polymers which combine the advantages of
well-defined supramolecular coordination complexes and
polymeric materials, though either hierarchical self-assembly
or postassembly polymerization.31−39 Motivated by our
previous studies on discrete metallacycles40 and phosphate
binding,41,42 we envisioned that a metallacyclic scaffold with
well-defined shape and size may provide an ideal platform to
construct polymers that can work as affinity reagents for the
recognition of PPs. The abundance of reactive sites on
polymeric scaffolds may enhance binding amount and provides
multipoint attachment between the target and the matrix,
which thus provides an opportunity to realize highly selective
and tunable capture of PPs.
INTRODUCTION
■
Anion recognition and extraction have received considerable
attention during the last few decades since anionic species play
many important roles in a number of areas including biology,
pharmacy, industry, and environmental sciences.1,2 To date, a
wide variety of functional groups and molecules have been
exploited to selectively recognize, respond to, or sense anionic
species.3−7 Recent results indicate that polymeric systems with
bona fide anion recognition features can offer special
advantages for the development of smart materials with
applications in anion recognition and extraction.8−11 However,
comprehensive analysis and identification of anionic bio-
molecules such as phosphorylated peptides (PPs) remain a
challenge owing to anions’ high hydration and PPs’ low
abundance as well as significant signal suppression by
nonphosphorylated peptides.12−14 Anionic biomolecules such
as PPs are closely associated with a number of human diseases,
including Alzheimer’s disease and cancer.15−17 Thus, the
development of supramolecular materials that can controllably
bind and release PPs under aqueous conditions has numerous
potential bioanalytical applications.
In this paper, we report a new kind of two-dimensional (2D)
covalently linked metallacycle-cored polymer (polymer 5) by
combining coordination-driven self-assembly and postassembly
One of the most successful approaches to date for the
recognition of phosphate derivate species in water is the use of
coordination complexes, inspired by the fact that metal cations
are commonly found in the binding sites of phosphate-binding
proteins.18,19 Over the past few decades, discrete supra-
molecular coordination complexes (SCCs) with well-defined
size, shape, and geometry have been widely constructed
Received: December 13, 2020
Published: May 27, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. (a) Schematic representation of free-standing 2D polymer formation by combining coordination-driven self-assembly and postassembly
1
reaction from simple organic ligands. (b) Partial H NMR spectra (in DMSO-d6, 400 MHz, 298 K) of 120° dipyridyl donor 1, hexagonal
metallacycle 3, supramolecular polymer 5, and methylene diphenyldiisocyanate 4.
Figure 2. (a) TEM image of supramolecular polymer 5, showing a 2D crumpled sheet image. The inset is a photograph of a polymer 5 dispersion
in DMSO (∼0.1 mg mL−1), showing the Tyndall effect. (b) TEM image of polymer 5 showing the coexistence of film-like structures with different
́
thickness. (c) Higher-magnification HR-TEM image of a polymer 5 sheet with moire patterns. (d) Higher-magnification HR-TEM image with the
corresponding FFT pattern, displaying a typical hexagonal atomic arrangement. (e) Perspective view of ScanAsyst mode AFM images by using the
ScanAsyst-Fluid+ probe and corresponding height profiles of a sample prepared by drop coating a suspension of polymer 5 obtained after a prolong
physical exfoliation by sonication in CH2Cl2 onto a mica sheet. (f) Histogram for statistics of the layer thickness for possible monolayers based on
103 different sites in AFM images. (g) HAADF-STEM image and corresponding EDX-mapping images (h) and SAED pattern (i) of polymer 5. (j)
Pictorial representation of layered films of polymer 5, in which each metallacycle has positive charges on both ends. Units are shown in a stick mode
with each layer in a different color and counterions omitted for clarity.
reaction. The supramolecular polymer contains both H-
bonding-based (urea) and electrostatic interaction-based
(metallacycle) phosphate recognition units, facilitating strong
and highly specific binding. Polymer 5 displayed a unique 2D
morphology that has binding sites at both surfaces, permitting
fast reactions with PPs, while nonphosphopeptides can be
removed by washing with water. Moreover, the dynamic nature
of metal−ligand bonds endows the polymer with excellent
tunability and controllability over interactions with substrates.
The unique stimuli-responsive properties facilitate rapid and
complete separation of the affinity material. Using this 2D
metallacycle-cored polymer 5 as an affinity material, we have
quantitatively identified different synthetic standard peptides,
demonstrating high enrichment capacity (up to 165 mg/g) and
recoverability (>65% for all standard peptides). Additionally,
the 2D metallacycle-cored polymer 5 is generally applicable to
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the analysis of PPs in complex protein mixtures, as well as in
the tryptic digest of nonfat milk, demonstrating the validity of
this enrichment approach and illustrating its potential use in
proteomics applications.
flexible film structure with overlays, roll-ups (Figures 2a and
S15), or agglomerations (Figure S16) was formed.
Similar sheet-like structures were observed in atomic force
microscopy (AFM) measurements (Figures 2e and S17) when
spin coating DMSO solutions of polymer 5 (3 or 1 mg/mL)
onto mica surfaces. The existence of multilayer structures with
crossover and overlapping areas (Figures S18 and S19) or the
coexistence of film-like structures with different transparencies
(Figures 2b and S20) were also observed, thus supporting the
existence of 2D layered structures in bulk solution. A scanning
electron microscope (SEM) investigation further confirmed
the sheet-like structures (Figure S21). We then employed
energy dispersive X-ray spectroscopy (EDS) to characterize the
elemental distribution. The elemental ratios for C, N, Pt, P,
and S (Figure S22) were in accord with the composition
expected for polymer 5. The high-angle annular dark-field
scanning transmission electron microscopy (HAADF-STEM)
image further confirmed their membrane-like structure, as
shown in Figure 2g. Most importantly, the elemental mapping
images clearly disclosed a homogeneous distribution of C, Pt,
P, etc., throughout the entire layer (Figures 2h and S22),
verifying that these sheet structures were generated from
supramolecular polymer 5, other than adventitious impurities.
Some sheets and films exhibit a quite low contrast against the
background (Figures 2a and S18), suggesting that they are
extremely thin, possibly a monolayer, although their thickness
could not be estimated from TEM. Further evidence comes
from the observation of nanosheets with a thickness around 1.7
nm from AFM (Figure S23), matching the thickness expected
for a monolayer. Due to the high electron beam sensitivity, the
transparent single sheets visibly degraded (Figure S24) during
high-resolution TEM (HRTEM) measurements or selected
area electron diffraction (SAED) analysis. This has been
observed previously for ultrathin 2D materials.43 For the
thicker multilayer films that can withstand bombardment by
the electron beam, a layer-stacking morphology with atom-
cloth-like patterns was observed under HRTEM imaging
(Figures 2c and S25), and regular apparent light−dark lattice
fringes with an average lattice spacing of ∼0.23 nm (Figures 2d
and S26) were found in selected areas. This spacing matches
well with that expected for Pt-containing structures. The
hexagonal patterns as well as the hexagonal diffraction spots in
the corresponding fast Fourier transform (FFT) patterns of a
6-fold symmetry which were different samples (Figures 2d and
S26) indicate the existence of a hexagonal arrangement inside
RESULTS AND DISCUSSION
■
Design, Synthesis, and Characterization of 2D Metal-
lacycle-Cored Polymer. To accomplish the efficient
construction of a metallacycle-cored supramolecular polymer,
metallacycles with functional reactive sites should be both
simple and stable, and the reaction used to link the
supramolecular metallacycles should be mild and highly
efficient. By examining several metallacycles and chemical
reactions, a donor ligand 1, containing two pyridyl units for
metal coordination and an amino group for polymerization,
was identified (Figure 1a). The hexagonal metallacycle 3 was
prepared in quantitative yield by mixing the 120° dipyridyl
donor 1 with 120° platinum acceptor 2 in a 1:1 ratio (Figure
1a and Scheme S1). The quantitative formation of amine-
containing metallacycle 3 was confirmed by multinuclear (1H
and 31P) NMR (Figures 1b and S1, S3, and S4), 2D COSY and
1H−1H NOESY NMR spectroscopy (Figures S5 and S6), and
electrospray ionization time-of-flight mass spectrometry (ESI-
TOF-MS) (Figure S7).
Stirring of a mixture of metallacycle 3 with methylene
diphenyl diisocyanate 4 in tetrahydrofuran solution at room
temperature, followed by precipitation with methanol, gave
polymer 5 in 93% yield (Figure 1a and Scheme S2). Due to its
highly cross-linked chemical structure, polymer 5 is insoluble
in most solvents; however it can swell and finally dissolve in
DMSO and DMF. An obvious Tyndall effect (Figure 2a inset)
confirmed the dispersibility of polymer 5 in solution, which
implies its excellent processability. Multiple peaks observed in
both gel-permeation chromatography (GPC) and dynamic
light scattering (DLS) analysis (Figure S8) provided evidence
supporting a broad mass and size distribution of polymer 5
from tens up to hundreds of kDa. Typical absorption bands of
the stretching vibration of CO (1700 cm−1) and the
hydrogen bonded N−H stretching vibration (broad bond at
∼3351 cm−1) were observed in the FTIR spectrum of polymer
5 (Figure S9), providing support for the formation of the
polyurea material. The thermal decomposition temperature of
polymer 5 was around 330 °C, indicating its high thermal
stability (Figure S10). The 31P{1H} NMR spectrum of polymer
5 exhibits a broad singlet at 13.10 ppm, which was similar to
that of hexagonal metallacycle 3 (Figures S2 and S11),
implying the persistence of metallacyclic structures in supra-
́
the structure. The presence of moire fringes is evidence that
the layered structure adopts slight interlayer offset stacking that
has been observed with other layered 2D polymers and 2D
COFs.44 SAED of these multilayers (Figures 2i and S27) was
consistent with their polycrystalline, hexagonally ordered
structure.
1
molecular polymer 5. In the H NMR spectra of 5, the amine
protons shifted from 5.33 ppm to 8.51 ppm and 7.41 ppm,
respectively (Figures 1b and S2 and S12) because of the
formation of polyurea. The signals corresponding to pyridyl
moieties Hα and Hβ and the aromatic protons H1, H2, H3, and
H4 remained, indicating that the postassembly reaction does
not perturb the hexagonal metallacycle scaffold. 2D COSY and
1H−1H NOESY NMR spectroscopy (Figures S13 and S14)
also confirmed the entirety of the metallacycle scaffold in the
polyurea polymer.
The morphology of supramolecular polymer 5 was
investigated using transmission electron microscopy (TEM).
By dripping the DMSO solutions of polymer 5 directly onto a
TEM support, followed by freeze-drying under vacuum, a
For comparative studies, samples were also prepared by
means of the top-down sonication of the bulk solid of polymer
5. Physical exfoliation of polymer 5 from the corresponding
bulk solid was conducted by sonication in CH2Cl2. TEM
images revealed unrolled and stacked polymer films with
terraces appearing at the borders (Figure S28), indicating their
multilayer structures. Sheet-like structures (Figure S30) were
also found in a SEM investigation of polymer 5 exfoliation
dispersions. EDS elemental mapping analysis associated with a
TEM image (Figure S29) and SEM image (Figure S30), as
well as surface composition investigated with X-ray photo-
electron spectroscopy (XPS) (Figure S31), found the presence
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of elements that were the same as those of solution samples,
suggesting again the formation of 2D sheet-like structures from
polymer 5. The SAED pattern of polymer 5 solids (Figure
S34) displayed a series of polycrystalline rings with some
discrete spots, in accordance with the results obtained from a
polymer 5 solution sample. The wide-angle X-ray diffraction
(WAXD) profile of polymer 5 showed one broad peak
centered at approximately 2θ = 25° (Figure S36), suggesting
its low crystallinity. This is due to the disordered random
stacking nature of the freestanding sheets with few-layer
numbers. The small-angle X-ray diffraction (SAXD) patterns of
polymer 5 solids displayed diffraction peaks corresponding to
about 3.6 nm (Figure S36), in agreement with the theoretical
pore widths (Figure S38) calculated from the expected
metallacycle units of the polymers. Small pores with a diameter
of about 3.3 nm (Figure S35), which is characteristic of the
metallacycle pores, were also observed in TEM measurements,
confirming the formation of 2D polymers with the expected
honeycomb-like backbones.
upon addition of Bu4NCl, forming an irregular nanostructure.
The diameter of the sample of the polymer decreased in the
DLS experiment (Figure S42) upon addition of Bu4NCl,
providing further evidence in support of the stimuli-
responsiveness of this system.
In situ multinuclear NMR (1H and 31P) investigations of the
polymer provided evidence that this stimuli-responsive
phenomenon was a consequence of the halide-induced
disassembly and reassembly of the hexagonal metallacycles
(Figure S43). After the addition of Bu4NCl, the proton
resonances of hexagonal metallacycles disappeared, along with
an increase in resonances from free the ligand, indicating
complete disassembly of the hexagonal metallacycle. The
1
original resonances of 5 in the H and 31P NMR spectra were
restored upon the addition of AgOTf to the mixture,
demonstrating the quantitative reassembly of the polymer.
The stimuli-responsive properties of polymer 5 offered
flexibility in controlling the behavior of polymer 5 using
external stimuli (Figure 3a), which would further determine
differential binding toward anions and PPs (see below) and
make it possible to modulate adsorption and release of PPs
(Figure 3b).
HRTEM images of supramolecular polymer 5 solid
multilayer films from different areas showed fine lattice
́
structures and moire fringes (Figure S32). Moreover, periodic
́
linear moire patterns with a periodic distance of 1.7 nm were
observed (Figures 2c and S33), in agreement with periodic
distance along one direction of layered stacking, suggesting the
retention of some vertical stacking. The formation of extremely
thin layers by physical exfoliation was observable in AFM
measurements (Figures 2e and S37). Cross-sectional analysis
indicated that the films were very flat and uniform. A statistical
study of the monolayer thickness suggests a mean average
height of about 1.7 nm (Figure 2f), which is similar to that
predicted by calculation.
The design and synthesis of 2D polymers is a challenging
task,45 especially for preparing 2D polymers in homogeneous
solution.46−49 However, with this system, a free-standing,
single-to few-layer 2D polymer film was prepared without any
preorganization of building blocks on solid surfaces or
interfaces. The semiempirical method PM750 in the program
MOPAC201651 was used to optimize the structure of the
positively charged metallacycle during the formation of the
polymer. We calculated the binding energies of two metalla-
cycle rings in simple stacking models (a, b, and c in Figure
S38) to examine how the metallacycle assembles into the
polymer. The simulation showed that the interactions between
two metallacycle rings were negligible, especially when the
interlayer distance was larger than 3 nm, while weak repulsions
between them blocked the extension of the metallacycle along
its vertical axis (Figure S38). Therefore, computational
simulations suggested that the three reactive groups attached
to the vertex of a hexagonal metallacycle may prefer growth as
an extension of its network along the horizontal axes due to
electrostatic repulsion. Figure 2j shows the simulated polymer
in its 2D layered offset stacking, in which the positive charges
on its skeleton keep each polymer sheet separated from one
another (Figure S39). Based on the above findings, the positive
charges on the metallacycle might be one of the most
important factors controlling the formation of 2D structures.
Stimuli-Responsive Properties of 2D Metallacycle-
Cored Polymer 5. The 2D metallacycle-cored polymer 5 was
expected to display stimuli-responsive switchable properties
due to the dynamic nature of metal−ligand bonds. As detected
by both AFM (Figure S40) and TEM (Figure S41)
measurements, the regular planar network of 5 dispersed
Figure 3. Stimuli-responsive properties of 2D metallacycle-cored
polymer 5. (a) Schematic representation of stimuli-responsive
disassembly and reassembly of 2D supramolecular polymer 5 and
(b) the corresponding binding/release toward PPs modulated by
external stimuli.
Binding and Affinity Properties of 2D Metallacycle-
Cored Polymer 5 toward Anions and Standard PPs.
Given that polymer 5 is positively charged and contains urea
hydrogen bond donors, we explored whether this system could
bind anions in solution. We found that upon addition of one
equivalent (concentration calculated relative to the stoichiom-
etry of the dipyridine units) of TBAH2PO4, the polymer
precipitated from solution and its 1H NMR resonances became
undetectable (Figure S44). Meanwhile, chemical shifts were
observed in both 1H and 31P NMR spectra of TBAH2PO4 with
the addition of a small quantity of polymer 5 (Figure S45),
−
indicating affinity between 5 and H2PO4 . To further
investigate the anion binding properties of polymer 5, UV
titration experiments, a typical and widely adopted method for
calculating the association constant (Ka) in host−guest
chemistry, were conducted to evaluate the apparent binding
−
2−
affinity of 5 with various anionic guests (i.e., H2PO4 , HPO4
,
PO43−, HCO3 , and CH3COO−, Figures S46−S50). As shown
in Figure 4a, upon slow addition of PO4 into a solution of
−
3−
polymer 5 (10 μM, calculated based on the concentration of
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3−
Figure 4. (a) UV−vis titration of polymer 5 (1.0 × 10−5 mol·L−1, based on the dipyridine units) with PO4 in DMSO solution at 20 °C with
tetrabutylammonium (Bu4N+) as cations. The absorbance values from 320 nm were fitted globally to a 1:1 binding model (assuming one dipyridine
unit of polymer 5 as one host, inset figure), giving an apparent binding constant of 4.4 × 105 M−1. (b) Typical fluorescence spectra of N-terminal
fluorescein-labeled serine phosphorylated peptide 3SP (1.0 × 10−6 mol·L−1) upon addition of different equivalents of polymer 5 (concentrations
are calculated based on the dipyridine units) in aqueous solution at 20 °C and the fluorescent intensity change (inset figure) of peptide hosts upon
the additions of guests. [L]/[PP] is an abbreviation of the molar ratio of guest to host. (c) Amino acid sequences of serine phosphorylated peptides
1SP−4SP and nonphosphorylated peptides NP1 and NP2 studied in this work. (d) Comparison of apparent Ka of polymer 5, metallacycle 3, unit 6,
and ligand 1 with various peptides. Data are from n = 3 independent experiments and are presented as mean s.e.m. (e) AFM images before and
after (f) treatment with serine monophosphorylated peptides 1SP.
dipyridine units within the polymer), a gradual decrease in
absorption intensity at approximately 320 nm was observed.
Polymer 5 was found to bind hydrogen phosphates and
phosphate strongly and selectively. The apparent association
constant of 5 with phosphate (Ka = 4.4 × 105 M−1) was about
54 times higher than that with acetate (Ka = 8.1 × 103 M−1)
(Table S1), demonstrating the phosphate selectivity of the
polymer. Anion complexation studies were also conducted
with metallacycle 3 (Figures S51−S55). Interestingly, affinities
for the tested anions with metallacycle 3 were observed
comparable to those with polymer 5 (Figure S57 and Table
S1), showing that the metallacycle skeleton is critical for
binding anionic guests and that the electrostatic interactions
may be the main driving force for anion complexation in these
systems. To prove that the disassembly of the metallacycle
backbone will result in a loss of binding affinity toward
phosphate, the phosphate affinity of ligand 1 was also studied
in a UV/vis titration experiment. As expected, negligible
conducted with the four model peptides and different
compounds including polymer 5, metallacycle 3, and related
compounds ligand 1 and unit 6 (Figure S40). It was found that
the fluorescence of the model PPs was efficiently quenched
upon addition of polymer 5 (as an example, the titrations with
3SP are shown in Figure 4b). In all cases, the calculated
apparent association constants between polymer 5 and PPs
were in the 105−106 M−1 range (Table S2), indicating that
polymer 5 was an ideal affinity candidate for binding PPs from
mono- to multiply charged PPs. For 1SP to 4SP, a similar turn-
off response was observed with metallacycle 3. By comparison,
the fluorescence intensity of the PPs did not show a significant
response toward ligand 1 and unit 6 (Figures S58−S61). These
results suggested again that the electrostatic interaction is the
main driving force in the interaction between polymer 5 and
PPs, and there is a significant difference in PPs’ binding affinity
between polymer 5 before and after external stimuli.
In complex samples such as cell lysates, several other protein
functional groups could compete for affinity enrichment.
Therefore, we performed control experiments in which
nonphosphorylated peptide with identical amino acid
sequences NP1 and another acidic peptide NP2 with Ser
residues substituted by Asp were used (Figure 4c). Under the
same conditions, for both NP1 and NP2, only slight decreases
in fluorescence intensity were observed (Figures S62 and S63),
with the calculated association constants being much lower
than those of the PPs (Figure 4d and Table S2), thus
indicating satisfactory discrimination ability of the polymer
between PPs and NPs.
−
absorption change was observed upon the addition of H2PO4
into the solution of ligand 1 (Figure S56), indicating that
ligand 1 display no capacity for binding phosphate. The very
different binding capacity of polymer 5 and ligand 1 toward
phosphate makes it possible to modulate the affinity of
polymer 5 using external stimuli to control polymer formation.
Four N-terminal fluorescein-labeled model peptides with
identical amino acid sequences differing only in the number of
phosphate groups (serine mono-, di-, tri-, and tetra-PPs,
abbreviated as 1SP−4SP, Figure 4c) were prepared to study
the binding affinity of polymer 5 for PPs. Fluorescence titration
experiments, in which Ka values were elucidated according to
intensity changes in the maximum emission peak, were
AFM measurements were then conducted to directly
visualize the interaction between the PPs and polymer 5. As
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shown in Figure 4e,f, upon adding a solution of 1SP followed
by washing with water, the polymer film expanded with the
morphology changing from thin film to thick cross-linked fiber.
No 1SP was retained on the surface of the blank substrate
(without the coating of polymer) after washing (Figure S64).
Consistent morphological changes were observed when the
polymer film was treated with other multiply charged PPs from
2SP to 4SP (Figures S65−S67). Under the same conditions,
no obvious change of film morphology and thickness was
observed upon interaction with nonphosphorylated peptides
NP1 and NP2 (Figures S68 and S69). These results further
confirmed satisfactory selectivity and high adsorption capaci-
ties of polymer 5 toward PPs.
Theoretical calculations of the possible binding modes and
binding energies between polymer 5 (metallacycle 3 was
considered as the monomer unit of polymer 5) and different
model peptides suggested that the skew-crossing mode and
skew-clinging mode are the dominant modes of interaction
(Figure S70 and Table S3). The calculated binding energy of
1SP was slightly larger than those of control model peptides
Asp-P and Glu-P, in which the phosphorylated serine group
was replaced by two other common negatively charged amino
acid Asp or Glu groups, respectively (Figure S71 and Table
S4). This result demonstrated the discrimination ability of the
polymer between PPs and other low isoelectric point peptides.
The binding energy increased with the number of phosphate
groups from 1SP to 4SP (Figures S72−S74 and Table S5).
Strategy for the Enrichment of PPs using 2D Polymer
5. The above results illustrate the potential of the newly
designed 2D supramolecular polymer 5 to capture PPs. To
validate this point, we evaluated the PP enrichment and
separation properties of polymer 5 using a phase extraction
method with 1SP as a model phosphoprotein sample. After
adding one equivalent of polymer 5 (Figure 5a), the green
emission of the 1SP solution decreased (91% quench) and
subsequently recovered (90% recovery) upon the addition of
Bu4NCl (Figure S75), indicating the effective capture and
release of 1SP. As shown in Figure 5b, the clear solution of 1SP
immediately becomes a milky, light yellow suspension after
addition of polymer 5 due to the interaction between polymer
5 and 1SP and the low solubility of polymer 5 in water. The
yellow polymer precipitates with a bulky fibrous morphology
(Figure S76) and can be easily collected by centrifugation. It is
worth highlighting that 1SP cannot be precipitated out of
solution by the interaction between 1SP and macrocycle 3,
although macrocycle 3 displayed affinity and selectivity toward
phosphorylated peptides similar to polymer 5, indicating the
superiority of polymer 5 as a PP affinity material. The high
binding affinity of polymer 5 with PPs makes it simple to
remove NPs that interfere with the analysis by simple washing
with water. The remarkable difference in binding affinity of
polymer 5 and ligand 1 with PPs enables the efficient isolation
of PPs and subsequent MS detection. Thus, as illustrated in
Figure 5c, an efficient protocol via a two-step elution
procedure under mild operating conditions was developed
for the enrichment and isolation of PPs.
Figure 5. Enrichment and separation of PPs in model protein
samples. (a) Emission intensity of 1SP before and after capture of
polymer 5. Inset is the photograph under UV. (b) Dispersity analysis
of 1SP before (left) and after (middle) adding of polymer 5 and after
gentle centrifugation (right). (c) Schematic illustration of PPs’
enrichment strategy using polymer 5. (d) MALDI-TOF mass spectra
of α-casein tryptic digest (1 pmol). Direct analysis (top) after
#
enrichment by polymer 5 (bottom, *phosphopeptide; dephosphory-
lated peptide). Detailed information about peptide sequences and
phosphorylation sites is shown in Supporting Information Table S6.
(e) MALDI-TOF mass spectra of tryptic digests of α-casein and BSA
at molar ratios of 1:10. Direct analysis (top) after enrichment by
#
polymer 5 (bottom, *phosphopeptide; dephosphorylated peptide).
severely suppressed by those of nonphosphorylated peptides.
Nevertheless, after the treatment by the polymer 5 as affinity
material, most of the nonphosphorylated peptides had been
removed and nine monophosphopeptides and 13 multi-
phosphopeptides (Figure 5d (bottom) and Table S6) were
observed after enrichment. As a control, only one phosphopep-
tide signal was detected after treatment by metallacycle 3, and
no signal could be detected after treatment with unit 6 or
ligand 1 (Figure S81). Moreover, fewer phosphopeptide signals
were detected after enrichment when using commercially
available titanium dioxide as an affinity material (Figure S77).
The data from three independent experiments show the high
degree of reproducibility of the polymer enrichment method
(Figure S79).
To further simulate the complex physiological environment,
semicomplex samples including tryptic digests of α-casein
mixed with different molar ratios (i.e., 1:10, 1:20, 1:100, and
1:500) of bovine serum albumin (BSA) as a model interfering
protein were tested. As shown in Figure 5e, when the molar
ratio of α-casein and BSA was 1:10, five monophosphopeptides
and five multiphosphopeptides were selectively enriched and
could be clearly detected. The satisfactory separation
capabilities of the polymer 5 toward PPs were still maintained
when the molar ratio of casein to BSA was increased to 1:100
and even to 1:500 (Figure S78). As a comparison, dioxide
PPs’ Enrichment from Model Protein Samples. We
applied the strategy first to the isolation of PPs from the tryptic
digest of a standard phosphoprotein (bovine α-casein). A
direct matrix-assisted laser desorption ionization (MALDI)
time-of-flight (TOF) mass spectrum of α-casein is illustrated in
Figure 5d (top). Signals of nonphosphorylated peaks
dominated the spectrum, and phosphopeptides signals were
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showed a preference for monophosphopeptides, leading to less
detectable multiphosphopeptides in the analysis of those
semicomplex samples (Figure S80).
phosphopeptides 2SP−4SP, the recoveries obtained using
polymer 5 were increased to about 88% with the increase of
charge number. The bound multiply charged phosphopeptides
could be disassociated from the polymer surface relatively
easily owing to the controllable binding behavior of polymer 5,
leading to high recoveries for PPs. Thus, a convenient
separation procedure, satisfactory recovery, high adsorption
capacities, and high detection sensitivity make polymer 5
suitable for enrichment and analysis of various phosphopep-
tides.
Application in Highly Specific Enrichment of PPs
from Nonfat Milk. Encouraged by the above results, to
further examine the selectivity and effectiveness of polymer 5
in the capture of low-abundance phosphopeptides from
complex samples, a tryptic digest mixture of nonfat milk,
which contains abundant proteins, including phosphoproteins
such as α-casein and β-casein, was prepared and tested.
Nonphosphopeptide signals dominated the mass spectrum
obtained from direct analysis of the tryptic digest of proteins in
nonfat milk, and no MS signal intensity of phosphopeptide was
detected (Figure S87). As shown in Figure 6c, after enrichment
with polymer 5, a total of 25 phosphopeptides including 16
monophosphopeptides and nine multiphosphopeptides, corre-
sponding to phosphopeptides of α-casein and β-casein, could
be identified, while fewer phosphopeptides could be detected
after enrichment with dioxide (Figure 6d). Detailed
information on the 25 phosphopeptides extracted from the
tryptic digest of nonfat bovine milk is provided in Table S7.
These results demonstrate the utility of this method and the
potential of polymer 5 to be used in the analysis of biological
samples in the future.
The concentrations of phosphopeptides bound to the
affinity materials are crucial for the identification of diagnostic
markers owing to their low abundance in the lysates of
biosamples. Therefore, the detection sensitivity for PPs was
measured by using low-concentration α-casein tryptic digest.
For polymer 5, two phosphopeptides were detected with an S/
N ratio of 8 and 4 for 2 fmol of α-casein tryptic digests (Figure
S82). The lower detection limit of polymer 5 may be attributed
to the high amount of immobilized PPs and the strong binding
interaction between phosphopeptides and polymer 5. This
result indicates that the polymer 5 has a high detection
sensitivity for phosphopeptides.
The enrichment capacity of polymer 5 toward PPs was then
investigated using standard peptides 1SP−4SP. Different
amounts of SPs in a fixed volume were incubated with a
fixed amount of polymer material until maximal loading was
reached. As illustrated in Figures 6a and S83, the adsorption
The potential of supramolecular polymers as affinity
materials to specifically and comprehensively isolate PPs
from a complex sample has never been evaluated previously.
Although the use of supramolecular coordination complexes as
sensors, catalysts, and biomedicines has been widely
reported,31−39 this is the first report on the application of
these systems in proteomics. The utility of soluble supra-
molecular polymers allows for fast homogeneous interaction
with PPs under mild conditions, which may be beneficial to
analysis of unstable PPs. In this study, the 2D supramolecular
polymer 5 was used to capture PPs owing to its morphology
and solubility, as well as controllable affinity upon external
stimuli. Our two-step approach based on polymer 5 enriching
PPs results in comparable performance to those of other
reported two-dimensional materials (Table S8).53,54 It is worth
highlighting that a number of TiO2- and IMAC-based
protocols have been highly optimized over the years, enabling
very high specificity and sensitivity for PP enrichment and MS
analysis.55,56 In contrast, despite the relatively immature status
of enrichment workflows employing our supramolecular
polymeric materials, results obtained thus far are comparable
with those from optimized methods employing TiO2 micro-
columns with similar detection strategies (MALDI-TOF).57
This indicates the scope for high performance of our material,
particularly with continued development of enrichment
workflows. We envision that further optimization of enrich-
ment protocols employing our polymeric material and the
combination of these with high performance detection
platforms will result in enhanced enrichment results.
Considering the potential for mild elution conditions by
decomposition of the polymer, we also envision a possible role
for the material in the analysis of acid-labile noncanonical
Figure 6. Adsorption capacities, recovery, and specific enrichment of
PPs from nonfat milk. (a) Comparison of adsorption capacities of
polymer 5 (black columns) and commercially available TiO2 (red
columns) toward 1SP−4SP. (b) Comparison of recovery of polymer 5
and commercially available TiO2 based enrichment methods toward
1SP−4SP, obtained from three parallel MS measurements. (c)
MALDI-TOF mass spectra of tryptic digests of the nonfat milk
after enrichment by polymer 5 (red, α-casein; blue, β-casein,
#dephosphorylated peptide) and commercially available TiO2 (d).
capacity of the material increased substantially with the
number of phosphates in the SPs. In comparison, titanium
dioxide displayed much weaker and unbiased adsorption
capacities52 toward various SPs (Figure S84). For example,
the enrichment capacity of polymer 5 toward 4SP was about
165 mg/g, nearly 10 times higher than that of titanium dioxide
(17 mg/g).52 The improved adsorption capacities of polymer 5
may be attributed to the unrestricted mass transfer and
improved accessibility of SPs toward the binding sites in the
homogeneous reaction-based enrichment.
The enrichment recovery of phosphopeptides (defined as
the ratio of released PPs to the total PPs, involved in both
binding and releasing processes of the PPs) was investigated
using nonphosphorylated peptides as a control. Fixed amounts
of standard phosphopeptides 1SP to 4SP were treated with
polymer 5, and the eluate was mixed with the same amount of
NP1 or NP2 (Figures S85−S86). As shown in Figure 6b, the
enrichment recovery of monophosphopeptide 1SP from
polymer 5 was about 65%. As for multiply charged
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phosphorylation (e.g., histidine phosphorylation), which is
particularly widespread in prokaryotes.58
Fan-Fan Zhu − Shanghai Key Laboratory of Green Chemistry
and Chemical Processes, Chang-Kung Chuang Institute,
School of Chemistry and Molecular Engineering, East China
Normal University, Shanghai 200062, China
Xu-Qing Wang − Shanghai Key Laboratory of Green
Chemistry and Chemical Processes, Chang-Kung Chuang
Institute, School of Chemistry and Molecular Engineering,
East China Normal University, Shanghai 200062, China
Xiang Wang − State Key Laboratory for Modification of
Chemical Fibers and Polymer Materials & College of
Materials Science and Engineering, Donghua University,
Shanghai 201620, China
CONCLUSIONS
■
In summary, by linking the hexagonal metallacycle 3 via a
reaction between amine and isocyanate, a free-standing,
metallacycle cross-linked, single-monomer-thick 2D stimuli-
responsive supramolecular polymer in solution was successfully
prepared. The multiple positive charges on the metallacycle
skeleton can keep each individual polymer sheet separated
from one another by electrostatic repulsion during synthesis,
resulting in a robust and readily transferable 2D metallacycle-
cored supramolecular polymer 5. Furthermore, the metalla-
cycle backbone endows the resultant polymer with a high
propensity for binding phosphate-containing anions and
peptides. The different binding capacities of polymer 5 toward
PPs before and after stimuli allow it to be used as a tunable and
specific affinity material for the enrichment of phosphopep-
tides. High enrichment capacities, detection sensitivity, and
excellent recovery are achieved with diverse PPs. Polymer 5
may also be used to identify low-abundance phosphopeptides
from complex biological samples. We believe that this work
provides the basis for the rational design of next-generation
affinity materials for comprehensive phosphoproteome re-
search based upon supramolecular structures.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c12904
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
L.-J.C., S.J.H., and P.A.G. acknowledge and pay respect to the
Gadigal people of the Eora Nation, the traditional owners of
the land on which we research, teach, and collaborate at the
University of Sydney. P.A.G. thanks the University of Sydney
and the Australian Research Council (DP180100612) for
funding. H.-B.Y. acknowledges the financial support of NSFC/
China (No. 21625202), Innovation Program of Shanghai
Municipal Education Commission (No. 2019-01-07-00-05-
E00012), and the Program for Changjiang Scholars and
Innovative Research Team in University. J.W. thanks the
Austrian Science Fund (FWF) Project M2709.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c12904.
■
sı
Experimental section, synthetic overview, a full set of
characterization data, and a complete set of binding and
enrichment studies (PDF)
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Corresponding Authors
Jin Wen − State Key Laboratory for Modification of Chemical
Fibers and Polymer Materials & College of Materials Science
and Engineering, Donghua University, Shanghai 201620,
China; Institute of Theoretical Chemistry, Faculty of Vienna,
University of Vienna, A-1090 Vienna, Austria;
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Hai-Bo Yang − Shanghai Key Laboratory of Green Chemistry
and Chemical Processes, Chang-Kung Chuang Institute,
School of Chemistry and Molecular Engineering, East China
Normal University, Shanghai 200062, China; orcid.org/
0000-0003-4926-1618; Email: hbyang@chem.ecnu.edu.cn
Philip A. Gale − School of Chemistry and The University of
Sydney Nano Institute (Sydney Nano), The University of
Sydney, Sydney, NSW 2006, Australia; orcid.org/0000-
0001-9751-4910; Email: philip.gale@sydney.edu.au
Authors
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Sean J. Humphrey − School of Life and Environmental
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