Design of pH-Responsive Polymer Monolith Based on Cyclodextrin

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Design of pH-responsive Polymer Monolith Based on Cyclodextrin Vesicle for Capture and Release of Myoglobin Haijiao Zheng, Xiqian Li, and Qiong Jia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18999 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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ACS Applied Materials & Interfaces

Design of pH-responsive polymer monolith based on cyclodextrin vesicle for capture and release of myoglobin

Haijiao Zheng a, Xiqian Li b, Qiong Jia a,

*

a

College of Chemistry, Jilin University, Changchun 130012, China

b

China-Japan Hospital of Jilin University, Changchun 130033, China

ABSTRACT: β-cyclodextrin vesicles (CDVs) were firstly introduced into polymer monolith to prepare pH-responsive adsorption material and used for capture and release of a cardiac biomarker, myoglobin (Myo). SH-CDV was decorated with adamantane modified SH-octapeptide to enhance the encapsulation and release rates of Myo. Afterwards, SH-CDV was introduced into polymer monolith via click reaction to produce a pH-responsive monolith. Combining with mass spectrometry detection, the CDV-based pH-responsive monolith was used for enrichment of Myo glycopeptides from mixture of glycopeptides and nonglycoprotein (BSA) tryptsin digests reach up to 1:10000. A limit of detection of 0.1 fmol was obtained for Myo glycopeptides in blood sample, indicating the high sensitivity of the method. The prepared CDV-based hybrid monolith demonstrated itself to be a promising material for capture of glycoproteins in complex samples, which provides an efficient strategy for the identification and discovery of biomarkers of acute myocardial infarction.

KEYWORDS:

pH-Responsive,

Cyclodextrin

vesicle,

spectrometry

*

Corresponding author. E-mail address: [email protected] (Q. Jia). 1

ACS Paragon Plus Environment

Monolith,

Myoglobin,

Mass

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. INTRODUCTION

Over the past few decades, scientists have made great efforts on the development of controllable vesicle system assembly because of its hollow and enclosed structures which endow it to be responsive to electro-, thermo-, UV-, pH-, or chemo- stimuli.1-5 Practical methods for preparing vesicles are focused on building surfactants, lipid molecules, amphiphiles, and so on.6,7 Among the diverse blocks, amphiphilic macrocycle vesicles are particularly attractive, which have achieved various applications such as diagnostics, drug/gene delivery, or separation sciences.8-13 In general, amphiphilic macrocycle vesicles possess topological structures due to host-guest interactions, which endow them with the self-selectivity to environmental responsiveness.14 For instance, Huang et al. developed amphiphilic pillar[5]arene pH-responsive vesicles and used to encapsulate TNT based on donor-acceptor interactions, providing a new idea for the application of vesicles to the fields of drug delivery and separation science.15 Currently, water surroundings are usually preferred for amphiphilic macrocycle vesicles since they are mainly used for drug delivery.16 It is still a great challenge for vesicles when placed in complex matrix environments. On the other hand, the repeated usage of vesicles is a prerequisite for their real applications, which is determined by the reverse process, i.e. disassembly of the assembled molecules.17 Functionalization of vesicles according to their reversible and repeatable mechanism can be an alternative to enhance the structure of the assemblies.18 In addition, functionalized vesicles can not only improve the assembly efficiency but also possess rich chemical sites, which benefit their applications to wider fields. As a typical natural cyclic oligosaccharide, β-cyclodextrin (β-CD) has hydrophobic central cavity and hydrophilic outer surface. β-CD is a competitive candidate for the application in various aspects including nanomaterials, molecular recognition, supramolecular polymers.19 2


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β-CD-based vesicles (CDV) have attracted much attention due to their unprecedented properties, especially stimuli-responsiveness.20 The outer and inner surfaces of CDV are covered with β-CD, therefore, it can enhance the binding affinity and selectivity for targets because of its simultaneous capture of multiple ligands.21 Furthermore, β-CD not only can combine the hydrophilic and hydrophobic moieties, but also can be regarded as a hydrophilic group because of the rich polar hydroxyl groups at its rim.22 CDV may act as a promising candidate in the field of adsorption since it exhibits binding capacity for various targets. In our previous work, we incorporated β-CD into polymer monolithic substrate materials and applied to the efficient enrichment of glycoproteins.23,24 The implications of the results stimulated us to develop β-CD to construct diverse pretreatment materials such as CDV-based adsorbents. Polymer monolithic substrate materials, commonly possessing high permeability, loading capacity, and separation efficiency, have been widely reported and applied in separation science of proteomics.25,26 Responsive monoliths are developed mostly based on N-isopropylacrylamide monomer, endowing the monoliths with thermo-sensativity.27,28 As can be expected, introducing CDV into monoliths can not only provide stimuli-responsiveness, but also improve the affinity with the targets. However, monoliths based on CDV have never been reported. As a glycoprotein, myoglobin (Myo) is regarded as a useful cardiac biomarkers for early detection of acute myocardial infarction, so sensitive and efficient detection of Myo is of great essential.29,30 In the present work, a functionalized CDV-based polymer monolith was firstly synthesized and applied to the enrichment of Myo. SH-CDV was grafted onto mesoporous poly(glycidyl methacrylate-pentaerythritol triacrylate) (poly(GMA-PETA)) monolith using in situ polymerization. The morphology and properties of CDV and CDV-based monolith were determined by scanning electron microscopy, transmission electron microscopy, dynamic light scattering, small-angle X-ray scattering, contact angles 3



analysis, thermogravimetric analysis, and ultraviolet-visible spectroscopy. SH-CDV existed as a sphere at pH 7.4 but changed to be a fiber at pH 5.0, by which the controllable enrichment of Myo was achieved. Encapsulation efficiency, loading capacity, and release rate of SH-CDV and dynamic binding capacity of poly(GMA-PETA-CDV) monolith under different pH values were investigated. Moreover, the efficiency of the CDV-based polymer monolith was evaluated by the enrichment of Myo from human blood samples. The present work is the first attempt of developing a hybrid material based on CDV and application to complex non-aqueous biological surroundings.

2. EXPERIMENTAL SECTION 2.1. Synthesis of SH-CDV. The host-guest synthesis of CDV was described in a previous literature18 with a little change and briefly illustrated in Figure 1. Firstly, β-CD 1 was dissolved in CHCl3 followed by evaporated using a rotary evaporator and dried in vacuum for 1 h to prepare unilamellar bilayer vesicles. The obtained thin film of 1 was hydrated with PBS and vortexed extensively at room temperature to provide a 1 mM turbid aqueous solution of 1. After sonication under heating for 30 min, a clear solution was obtained. The size of the vesicles was set around 90 nm by extrusion through a polycarbonate membrane. Secondly, octapeptide was dissolved in a mixture of 0.1 mM EDTA and 0.15 M sodium borate at pH 8.0 containing Traut’s reagent. The mixture was shaken at 550 rpm for 1.5 h at 4 oC in the dark. The mole ratio of Traut’s reagent to octapeptide was 1.5/1. Next, PBS (pH 7.0) was used to exchange the solution by centrifugation to obtain SH-octapeptide 2. Thereafter, 2 was modified with 1-adamantanecarboxylic acid to prepare Ad-octapeptide -SH 3. An appropriate amount of 3 (typically 0.5 mM solution in PBS) was added into the vesicles (typically 1.0 mM solution in PBS) when pH of the mixture was set at 5.0 adjusted with 1 M HCl.18 Finally, after purification and vacuum drying, the final product 4


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CDV-Ad-(leu-glu)4-SH (SH-CDV) was obtained. The prepared SH-CDV was characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), Fourier-transform infrared (FT-IR), and nuclear magnetic resonance (NMR) determinations.

Figure 1

2.2. Myo glycopeptide enrichment. The synthesis process of poly(GMA-PETA-CDV) monolithic column was listed in Supporting Information. In the microextraction step, the column was firstly equilibrated with 0.5 mL MeOH and 0.5 mL loading buffer (pH 5.0). Afterwards, 1.0 mL sample solution (pH 7.4) passed the column (10 µL min−1), after which the above loading buffer was pushed through the column at the flow rate of 10 µL min−1. Finally, 30 µL ACN/H2O/FA (70:29.9:0.1, v/v/v, pH 5.0) was used as the eluent to release the glycopeptides (Figure 2). The first 10 µL eluent was directed for MS analysis because it could elute more than 95% glycopeptides from the monolithic column. Deglycosylation experiments of N-glycopeptides by PNGase F were also investigated. The eluent obtained after the enrichment step was dissolved in 50 µL 50 mM PBS (pH 7.4), after which PNGase F was added into the solution followed by incubated overnight at 37 °C 5



for deglycosylation. Next, the solution was heated to 100 °C for 10 min to stop the reaction. The obtained solution was introduced into the MS inlet for subsequent analysis.31

Figure 2

3. RESULTS AND DISCUSSION 3.1. Morphology and structure properties of SH-CDV. Firstly, the intermediate 1 was characterized by

13

C NMR and 1H NMR. Results were shown in Figure S1. Obviously, the

peaks at 1.198, 3.395, and 39.222 ppm confirmed that 1 was successfully synthesized. The as-prepared SH-CDV was characterized using γ, CMC, DLS, and SAXS determinations and indicated in Supporting Information (Figures S2, S3). Figure 3a showed TEM image of SH-CDV spheres, revealing that the colour of the peripheries and centers was different which was characteristic of hollow spheres. In Figure 3b, SEM image of the spheres was shown, indicating that the spheres aggregated with an average diameter of ∼90 nm which was in agreement with the DLS, SAXS, and TEM results mentioned above. It could also be observed from Figure 3b that the wall thickness of a fractured SH-CDV sphere was about 8 nm. pH responsibility and reversibility of SH-CDV. A schematic illustration of the reversible morphological change from vesicles to fibers was shown in Figure 2. As shown in Figures 6


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3b, 3c, the aggregate diameter of ~90 nm decreased dramatically to 8 nm when pH was change from 7.4 to 5.0. In the meantime, the aggregates maintained a narrow size distribution at the state of fibers (Figures 3c, 3d). The reversibility was achieved by adjusting the pH value back to 7.4, i.e., spherical vesicles could be seen again as shown in the SEM image in Figures 3e, 3f. Furthermore, the volumes of sphere and tube (Vs and Vt) could be calculated as Vs = (4/3)πrs3 and Vt = πrt2lt, respectively.18 As a result, the interior volume of SH-CDV was about 6 times that of the fiber tube. Therefore, it can be expected that SH-CDV can encapsulate hydrophilic guests within the interiors at pH 7.4 and release them in response to a decrease of pH.

7



Figure 3

FT-IR spectra were determined to verify the pH-responsive structure transitions.15 Figure S4 indicated the comparison of 1 and fiber tubes assembled from 1. It could be found that the carbonyl bands and N−H stretching of 1 appeared at 1680 and 3400 cm−1 while shifted to 3396 and 1674 cm−1 in the fiber tubes. The decrease in the wavenumber suggested an enhancement of hydrogen bonding in the fiber tubes. In addition, the antisymmetric and symmetric stretching vibrations of CH2 in the fiber tubes existed at 2920 and 2850 cm−1 while 8


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the corresponding peaks for 1 were at 2926 and 2855 cm−1, implying an increase in the number of van der Waals interactions between adjacent alkyl chains in the fiber tubes.31 Since the transition between amphiphilic vesicles and fibers could be achieved by adjusting pH values, controllable encapsulation and release of hydrophilic guest molecules could be envisioned. With this in mind, Myo was encapsulated in the vesicles as a model hydrophilic guest. Generally, an increase in UV absorbance appeared when Myo was released from the interiors of SH-CDV and the free Myo in solution was dequenched. UV spectra of Myo encapsulated in 1.0 × 10−4 M vesicular solution at different pH values were shown in Figure S5. At pH 7.4, Myo was encapsulated in SH-CDV with an encapsulation efficiency of 89% accompanied by the loading efficiency of 75%. In addition, the entrapped Myo did not leak over a period of 24 h, indicating the stability of these vesicles. However, significant release of the encapsulated Myo was achieved at pH 5.0 with a release rate of 95%. These experimental data further verified the pH-responsive vesicle-to-fiber tube transition.

3.2.

pH-Responsive

poly(GMA-PETA-CDV)

monolith

construction.

The

experimental parameters in the synthesis process were optimized. In the present study, he proportions of monomer (GMA), cross-linker (PETA), and ternary porogen (PEG 6000 + PEG 20000 + H2O) were investigated. Results were listed in Table S1. Finally, Column 6 was selected as an optimum. The structural stability and mechanical property of the monolith were also studied since they are important for practical applications. The swelling ratios of monoliths in DMSO were also shown in Table S1. Results confirmed the favorable stability of monolith, which will play a dominant role in its practical applications. Next, SH-CDV amounts introduced into monolith were optimized. Increasing SH-CDV amounts could improve the extraction

9



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performance for Myo but lead to an increase in flow resistance. As an option, 50 mg SH-CDV was chosen to be introduced into the polymerization mixture. Thermal stabilities of poly(GMA-PETA) (inset) and poly(GMA-PETA-CDV) monoliths were measured with TG under a nitrogen atmosphere (Figure S6a). Compared with the thermograms of the former, the degradation curve of the latter demonstrated a three-stage process. The weight loss of 10% between 35 oC and 160 oC might be attributed to the damage of water vaporization and other volatile impurities. The third stage take place between 200 oC and 400 oC might be mainly due to the damage of SH-CDV and monolithic matrix. The third stage occurred at the temperature higher than 500 °C, illustrating a decomposition of the monolithic material. In Figure S6b, XPS results of poly(GMA-PETA) and poly(GMA-PETA-CDV) monoliths were indicated. The contents of N1p (408.5 eV) and O1s (532.9 eV) increased after the modification step, indicating that CDV was embedded into the monolith successfully. In addition, a new peak (S2p, 164.7 eV) appeared after introducing SH-CDV into the monolith, demonstrating that the click reaction occurred between poly(GMA-PETA) monolith and SH-CDV N2 adsorption-desorption measurements were conducted to characterize the porous structure of poly(GMA-PETA-CDV) monolith. As shown in Figure S6c (inset), Brunauer-Emment-Teller

(BET)

surface

areas

of

poly(GMA-PETA)

and

poly(GMA-PETA-CDV) monoliths were determined to be 57.4 and 135.7 m2 g−1, respectively. The hysteresis in the adsorption-desorption isotherm of poly(GMA-PETA-CDV) monolith implied the presence of mesoporosity. The sharp untrue and the absence of a saturation plateau at P/P0 of 0.9 MPa suggested the existence of macropores in the network. The isotherm exhibited a typical type IV curve with an H2-type hysteresis loop, implying that the mesopores were irregularly shaped in the dense network.32 Barrett-Joyner-Halenda (BJH) 10


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analysis indicated that the sizes of mesopores and macropores mainly concentrated in the range of 500–700 nm (Figure S6d). The results indicated the potential application of poly(GMA-PETA-CDV) monolith as a sample preconcentration material. The surface morphology and internal structure of poly(GMA-PETA-CDV) monolith at different pH values were determined by SEM and TEM (Figure 4). As a characteristic profile of porous material, homogeneous and multiple-pore structure morphology could be observed in Figures 4a, 4b. SEM and TEM images of poly(GMA-PETA-CDV) monolith at pH 7.4 (Figures 4a, 4c) showed that SH-CDV was successfully introduced into poly(GMA-PETA) monolith. Then, when pH decreased in 5.0, it could be clearly observed that a structural transformation appeared from vesicle to fiber tube (Figures 4b, 4d). To evaluate the impact of polymer matrix on the reversible transition from fibers to vesicles, the morphology of poly(GMA-PETA-CDV) monolith after adjusting the pH back to 7.4 was determined. It could be seen from Figure S7 that spherical vesicles could be observed again, indicating that polymer matrix had little influence on the reversible transition. In addition, TEM images revealed that they were uniformly distributed in amorphous three-dimensional mesoporous architecture, which not only favored the low back pressure of the monolith, but also benefitted the high enrichment ability of Myo glycopeptides.23 The results of back pressure experiments revealed that poly(GMA-PETA-CDV) monolith was able to sustain the back pressure of more than 240 bar in the flow rate range of 0.1-60 µL min−1 (Figure S8).

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Figure 4

Furthermore, the hydrophilicity of the monolith before and after SH-CDV modification was characterized by contact angles test. Compared with Figure 4e, after SH-CDV was introduced into the monolith, water film was nearly completely spread on the surface with CA distinctly decreased (Figure 4f). In addition, after poly(GMA-PETA-CDV) monolith was equilibrated in loading buffer (pH 5.0), CA was tested again, giving a value of 109.4o (Figure S9). These results demonstrated the improved hydrophilicity of poly(GMA-PETA-CDV) 12


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monolith at pH 7.4 and a contrary result at pH 5.0, which further proved that the SH-CDV based monolith compared favorably to poly(GMA-PETA) monolith toward hydrophilic target analytes. 3.3.

Applications

of

the

pH-response

monolith.

The

recognition

of

poly(GMA-PETA-CDV) monolith toward glycopeptides was mainly based on pH-responsive property of CDVs. In order to confirm the role of vesicle-to-fiber transition in the PMME process, effects of pH values of sample solution and eluent on the enrichment were investigated using tryptic digests of Myo (10 pmol). When sample pH was set at 7.4, the elution effects of ACN/H2O/FA (70:29.9:0.1, v/v/v, pH 5.0) and ACN/H2O/FA (70:29.9:0.1, v/v/v, pH 7.4) were compared and shown in Figures 5a, 5b. Figure 5b exhibited favorable elution ability at pH 5.0 rather than 7.4, which was because a decreased pH resulted vesicle-to-fiber transition and led to the release of Myo glycopeptides. On the other hand, effects of sample pH on the enrichment efficiency were investigated when ACN/H2O/FA (70:29.9:0.1, v/v/v, pH 5.0) was employed as the eluent. Compared with Figure 5b, much less Myo glycopeptides could be detected at sample pH of 5.0 (Figure 5c), demonstrating that poly(GMA-PETA-CDV) monolith could not adsorb the glycopeptides when SH-CDV was stay in fiber tube state.

Figure 5

13



Dynamic binding capacity. DBC is often used to describe the fraction of equilibrium binding capacity when all the binding sites are occupied.33 DBC of the monolith toward Myo was determined by a frontal spectrum method. BSA was eluted at the void time, whereas Myo was totally captured until the monolith was saturated. The breakthrough curves of poly(GMA-PETA-CDV) monolith were measured and shown in Figure S10. At sample pH 7.4, the breakthrough curve showed increasing signal between 10 mL and 14 mL, which was mainly because of the interaction between poly(GMA-PETA-CDV) monolith and Myo glycopeptides and consequently resulted in a kinetic adsorption process. DBC of the CDV-based monolith was approximately 35.5 mg g–1 at pH 7.4, suggesting the potential to use poly(GMA-PETA-CDV) monolith for preparative real applications of glycoprotein purification. In addition, DBC was calculated to be 0.5 mg g–1 at pH 5.0, further confirming that the encapsulation of Myo could be preferably achieved at pH 7.4 rather than pH 5.0. To investigate the enrichment ability of poly(GMA-PETA-CDV) monolith, 1 pmol tryptsin digests Myo were detected via direct injection and after the enrichment in the full scan mass spectra (m/z 800–3500), respectively. In Figure 6a, most peaks were raised from non-glycopeptides while glycopeptides were almost undetectable. The MADLI-MS spectra obtained after the elution step by poly(GMA-PETA) and poly(GMA-PETA-CDV) monoliths were shown in Figure 6b, 6c, respectively. It could be observed that the relative abundance of glycopeptides was increased and 15 glycopeptides could be found after enrichment. The enrichment efficiency of poly(GMA-PETA-CDV) monolith for Myo glycopeptides was further investigated. The eluent obtained after the enrichment with the developed monolith was treated with PNGase F for deglycosylation. It could be seen from Figure 6d that all the signals with m/z greater than 1200.0 disappeared. Only the deamidated peptides were observed, confirming that the signals in Figure 6b were glycopeptides.34 The amino

14


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sequences and proposed oligosaccharide compositions of Myo glycopeptides were listed in Table S2.35

Figure 6

To further evaluate the enrichment ability of poly(GMA-PETA-CDV) monolith for Myo glycopeptides, MS signals obtained from tryptsin digest mixture of BSA and Myo (10:1, 1000:1, and 10000:1, m/m) with and without the PMME pretreatment procedure were determined (Figure 7). Glycopeptides could be hardly detected without enrichment (Figures 7a, 7c, 7e), while the glycopeptides signals were significantly enhanced after enrichment by poly(GMA-PETA-CDV) monolith (Figures 7b, 7d, 7f). It was worthy to note that glycopeptides could be enriched even in the mixture containing an overwhelming amount of BSA (BSA:Myo of 10000:1) (Figure 7f). The results revealed that poly(GMA-PETA-CDV) monolith had high capacity in capturing glycopeptides from semi-complex sample, suggesting its great potential to enrich glycopeptides from a complex biological sample.

15



Figure 7

The developed poly(GMA-PETA-CDV) monolith was further applied to PMME-MS for the glycosylation sites profiling of human blood Myo. A series of diluted and post processing blank blood samples were spiked with Myo tryptic digests standard solutions to prepare working solutions in the concentration range of 0–5 pmol Myo. The total ionic signal intensity was linearly in the range from 0.005 to 5 pmol. 16


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The detection limit was investigated by loading Myo tryptic digests with different concentrations onto poly(GMA-PETA) and poly(GMA-PETA-CDV) monoliths. After enrichment by poly(GMA-PETA) monolith, only one signal of glycopeptide (T11) with low signal to-noise ratio (S/N) observed in the MS spectrum (Figure S11a). When diluting Myo digest to 1 fmol, 9 peaks of glycopeptides could be detected after enrichment by poly(GMA-PETA-CDV) monolith (Figure S11b). When further diluting Myo digest, 4 (T1, T4, T7, and T11, Figure S11c) and only 1 peak (T11, Figure S11d) could be observed for 0.1 fmol and 0.05 fmol Myo digests, respectively. As a consequence, the detection limits obtained by poly(GMA-PETA) and poly(GMA-PETA) monoliths, i.e., 10 fmol and 0.1 fmol with relative standard deviations (RSDs) below 10.0%. The stability of poly(GMA-PETA-CDV) monolith was investigated with spiked 1 pmol Myo tryptic digest in human blood sample. Results indicated that after the column was used for at least 90 times over one month (Figure S12). Myo glycopeptides could be still detected, implying that the monolith had good reusability. In addition, we studied the intra-batch and inter-batch reproducibilities for Myo glycopeptides enrichment of the monolithic columns, giving intra-batch and inter-batch reproducibilities of 2.3% and 3.5%, respectively. Consequently, high reproducibility of the preparation of monolithic columns was achieved. The comparison study about the differences between the developed PMME-MS strategy and other reported sample preparation procedures was also conducted. Results listed in Table S330,36-40 demonstrated that the LOD obtained by this method was lowest, implying that the present method had high sensitivity. Also, the LOD (0.1 fmol) is better than our previous results and some most recent developed CD-based materials for analysis of glycopeptides, most of which achieved the LOD 0.5–67 fmol (Table S423,24,29,41,42).

17



3.4. Real sample analysis. For 1 µL human blood samples, 166 N-glycosylated peptides could be identified attributing to 130 N-glycoproteins in three runs (Table S6, Figure S13), revealing its potential for large scale glycoproteomics analysis. Glycopeptides in undiluted blood samples were compared before and after enrichment by poly(GMA-PETA-CDV) monolith and then subjected to high resolution mass spectrometry analysis. Results were shown in Figure 8, indicating that Myo glycopeptides in blood samples after enrichment (Figure 8b) were more than those before enrichment (Figure 8a). A typical mass spectrum of the identified Myo peptides was shown in Figure 8c. A series of consecutive b and y ions in the figure demonstrated the high confidence of the identified Myo peptides.43 Furthermore, we observed distinguished pattern of fragment ions from Myo in the lower mass region of the spectrum. Xcalibur Qual Browser software was employed to calculate the molecular formulas of the daughter ions according to the accurate m/z values obtained by UHPLC-Orbitrap Fusion TMS determinations. In conclusion, although glycopeptides in human blood samples covered a wide dynamic range in abundance, the present monolith demonstrated its advantages of high enrichment efficiency of Myo glycopeptides and favorable feature for the applications in real samples.

18


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Figure 8

4. CONCLUSIONS

In summary, a pH-responsive CDV-based PMME-MS system was developed for high throughput interrogation of trace Myo glycopeptides in complex biological samples. The 19



structure and morphology of SH-CDV and SH-CDV-based monolith were characterized with NMR, FT-IR, TEM, SEM, SAXS, static surface tension, and DLS determinations. The prepared monolith was capable of recognizing the target glycopeptides in response to pH changes and exhibited a favorable binding kinetics. By combining with MALDI-MS detections, 15 glycopeptides from Myo digest were captured with the developed monolith. The developed method gave a linear range of 0.005–5 pmol for Myo glycopeptides with a regression coefficient greater than 0.9993, and LOD of Myo glycopeptides as low as 0.1 fmol was achieved. In addition, the SH-CDV-based monolith could capture Myo glycopeptides from Myo and BSA glycopeptides mixtures even with 10000 times mass interference. When used for the characterization of human blood samples, 166 intact glycopeptides from 130 glycoproteins were identified. Considering its simplicity and robustness, this PMME-MS strategy promises to be a potential tool for systematically analyzing protein glycosylation in other biological samples, such as tissue or cell line.

Acknowledgements

The project was supported by National Natural Science Foundation of China (21575049).

Supporting Information

Reagents and instrumentation; Sample preparation; Myo digestion; Characterization; MS identification; Database search and data analysis; CDV formation and characterization; Figures S1 ̶ 13 and Tables S1 ̶ 5.

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CAPTIONS

Figure 1. Synthesis of poly(GMA-PETA-CDV) monolith. Figure 2. pH reversible morphological change of SH-CDV and PMME process. Figure 3. (a) TEM images of SH-CDV; (b) SEM image of a ruptured vesicle; (c) TEM and (d) SEM images of fiber tubes at pH 5.0; (e) SEM image and (f) DLS result of SH-CDV after adjusting the pH back to 7.4. Figure 4. SEM and TEM images of poly(GMA-PETA-CDV) monolith: (a) and (b) SEM images at pH 7.4 and 5.0; (c) and (d) TEM images at pH 7.4 and 5.0; CA results of (e) poly(GMA-PETA) and (f) poly(GMA-PETA-CDV) monoliths (pH 7.4). Figure 5. MS spectra of 1 pmol Myo tryptic digests. Figure 6. MS spectra of 1 pmol Myo tryptic digests. (a) Direct analysis, (b) after enrichment by poly(GMA-PETA) monolith, (c) after enrichment by poly(GMA-PETA-CDV) monolith, and (d) after enrichment by poly(GMA-PETA-CDV) monolith and then deglycosylation by PNGase F. The peaks of glycopeptides or their fragments were marked. Figure 7. MALDI mass spectra of tryptic digest mixtures of Myo and BSA without (a, c, e) and with (b, d, f) enrichment by poly(GMA-PETA-CDV) monolith. Molar ratios of BSA to Myo were 10:1 (a, b), 1000:1 (c, d), and 10000:1 (e, f), respectively. The peaks of BSA peptides (yellow star) and Myo glycopeptides (blue star) were marked according to their mass charge ratios. Figure 8. MS spectra of Myo glycopeptides in human blood sample. (a) Before enrichment, (b) after enrichment by poly(GMA-PETA-CDV) monolith, and (c) a typical MS/MS spectrum of Myo peptides. The peaks of Myo glycopeptides were marked according to their mass charge ratios. 27



For Graphic Abstract

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Design of pH-Responsive Polymer Monolith Based on Cyclodextrin

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