Green synthesis of monolithic enzyme microreactor based on thiol-ene click reaction for enzymatic hydrolysis of protein

Journal Pre-proof

Green synthesis of monolithic enzyme microreactor based on thiol-ene click reaction for enzymatic hydrolysis of protein Xue Zhao , Pei-Ru Fan , Chun-E Mo , Yan-Ping Huang , Zhao-Sheng Liu PII: DOI: Reference:

S0021-9673(19)31025-8 https://doi.org/10.1016/j.chroma.2019.460618 CHROMA 460618

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

12 July 2019 25 September 2019 10 October 2019

Please cite this article as: Xue Zhao , Pei-Ru Fan , Chun-E Mo , Yan-Ping Huang , Zhao-Sheng Liu , Green synthesis of monolithic enzyme microreactor based on thiol-ene click reaction for enzymatic hydrolysis of protein, Journal of Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460618

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Highlights 

IMER based on deep eutectic solvent was prepared by " thiol-ene " click reaction.



Michaelis constants and maximum reaction rate were 2.1 mM and 0.028 μmol/min.



A rapid digestion of BSA on the IMER was 864 times faster than in-solution digestion.



1034 protein groups can be identified from protein extracts of rat liver by the IMER.

1

Green synthesis of monolithic enzyme microreactor based on thiol-ene click reaction for enzymatic hydrolysis of protein

Xue Zhao

Pei-Ru Fan

Chun-E Mo

Yan-Ping Huanga,*

Zhao-Sheng

Liua,*

Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China

Correspondence: Dr. Zhao-Sheng Liu Fax: +086-022-23536746 E-mail: [email protected] Correspondence: Prof. Yan-Ping Huang E-mail: [email protected]

KEYWORDS: Monolithic enzyme microreactor, Deep eutectic solvents, Room temperature ionic liquid, Click reaction

2

Abstract

In this study, a monolithic enzyme reactor based on a strategy of green synthesis was successfully prepared in a capillary with trypsin immobilized by " thiol-ene " click

reaction.

acid-co-ethylene

A

polymer

glycol

of

poly(butyl

dimethacrylate)

was

methacrylate-co-α-methacrylic prepared

in

a

mixture

of

1-butyl-3-methylimidazolium tetrafluoroborate and choline chloride/ethylene glycol as the support of enzyme reactor. After “thiol-ene” reaction was used for enzyme immobilization, the Michaelis constants and maximum reaction rate of the resulting immobilized enzyme reactors (IMER) were determined by capillary electrophoresis to be 2.1 mM and 0.028 μmol/min, respectively. The enzymatic hydrolysis of the enzyme reactor under different experimental conditions were investigated. A on-line digestion of bovine serum albumin (BSA) on the new IMER can be achieved within 50 s, up to 864 times faster than in-solution digestion (12 h). BSA can be well digested and the numbers of identified peptides were 73 with the coverage rates of 82.7%. The IMER was further used for the analysis of protein extracts from rat liver, and 1034 protein groups were identified. All these results demonstrated that such a click reaction based IMER would be of great prospect in the high throughput analysis for proteome with high confidence.

3

1. Introduction The large-scale and direct determination of cellular function at the protein level is in general dealt with proteomics [1,2]. At present, the commonly used technical route is Short-Gun technology (shotgun method) [3]. It involves in-solution digestion of all proteins and peptides separation of the enzymatically decomposed mixtures, followed by MS and/or tandem mass spectro-metric (MS/MS) analysis and protein identification by database search [4,5]. The traditional method of enzymatic hydrolysis in solution, however, has the problems of long enzymatic hydrolysis time, low analytical flux and self-hydrolysis, and is difficult to be used for microprotein sample analysis [6]. In shotgun proteomics approach, efficient digestion of proteins remains a key step for identification of protein successfully. To increase the proteomic workflow, one promising approach is to create a flow-through enzyme reactor permitting proteins pass through. Since the proteins are digested on-column, laborious manual steps involved with the off-line digestion can be avoided, eliminating sources of potential error. The immobilized enzyme reactor (IMER) based on organic monolithic column is good choice for this purpose since it often offer high enzyme-to-substrate ratio and a higher digestion efficiency compared to in-solution digestion due to high permeability of monolith [7-12]. However, the desired IMER in proteolysis remains to be developed since complex samples for IMER often cause a decrease in the enzyme activity. To obtain high enzyme activity as well as limited nonspecific adsorption, different immobilization protocols for stable IMERs have been explored [13-19]. In addition, digestion efficiency has been found

4

to depend on multiple factors, including the coupling chemistry affecting enzyme stability and activity, immobilization capacity, and nature and properties [20]. Because of effective generation of covalent links selectively under mild reaction conditions, " click chemistry" is as one of the most reliable approaches for covalent assembly of various molecules [21]. As a result, the approach has been introduced to prepare IMERs. For example, CuAAC is often used to prepare clickable silica monoliths [22]. However, this approach requires tedious time-consuming procedures with multiple steps. To address these problems, a clickable azido/alkynyl-silica hybrid monolithic column was made, which introduces reactive alkynyl or azido moieties with in situ poly-condensation of silane precursors via a simple “one-pot” approach [23]. However, the alkynes or azides groups must be firstly introduced, which is complicated with a risk of the decrease in enzyme activity. " Thiol-ene " reaction is one of the typical click chemical reactions [24,25], in which a thiyl radical is produced by an addition of radical source to an alkene double. By producing a carbon radical, a new hydrogen from another thiol can be abstracted and thus propagating the cycle. It is not necessary to add a catalyst during the reaction, and the reaction conditions are mild and rapid, and the product structure is easy to control. As a result, the immobilization of enzyme via thiol-ene chemistry is easier and simpler than previous methods as there are natural thiol groups in enzyme. The preparation of monolithic column involves mixing of one or more monomers, a crosslinker, as well as a porogen. In this case, porogen is a important reagent

and

by

changing

the

composition

5

of

porogens

used

or

the

monomer-to-porogen ratio, pore sizes can be optimized as required. Classic porogens are those volatile solvent (VOC) or their mixture [7-9]. Up to date, an increased emphasis on the “green” chemistry is frequently observed, which aims at eliminating or at least minimizing generated waste. In the design of a synthetic route, any attempt must comprehensively address these principles to meet green goals. Utilization of environmentally benign solvents, nontoxic chemicals, and renewable materials are important consideration in a green strategy. As a nonvolatile solvent of low vapor pressure, room temperature ionic liquids (RTILs) have attracted increasing interest in chemical research due to excellent solvation qualities [26]. Many interesting results have been produced in RTILs. However, RTILs have poor biodegradability and biocompatibility. As alternative RTILs, deep eutectic solvents (DESs) are developed to overcome the potential toxicity of RTILs [27], which are consisted of hydrogen bond acceptors and hydrogen bond donors. DESs were comparatively cheap and can be prepared conveniently compared with traditional RTILs. Some optional properties can be observed on DESs. For example, the interaction of DES with the functional monomer can improve the affinity and selectivity of the resulting molecularly imprinted polymer [28]. The combination of DESs and RTILs as porogen has also displayed advantage to prepare inorganic and organic hybrid monolith [29]. In view of facts above, a greener approach for the synthesis of monolithic IMER was proposed. In the present study, a green and non-volatile solvent, RTILs and DESs were used to prepare monolith as the supporter of IMER. A large amount of ethylene

6

glycol dimethacrylate (EDMA) used in the formulation provided the required double bond on the resulting monolith for thiol-ene click reaction. By using the residual double bonds on the monolithic column, the enzyme was immobilized with the thiol of trypsin, thereby avoiding secondary grafting on the monolithic column. This approach eliminates the modification step for the resulting monoliths. The activity of the enzyme reactor and the hydrolysis effect of the substrate under different reaction conditions were further evaluated. 2.1. Experimental 2.1 Materials and chemicals γ-Methacryloxypropyltrimethoxysilane (γ-MPS, 98%) was ordered from Acros (Geel,

Belgium).

Azobisisobutyronitile

(AIBN,

AR

grade)

and

Tris(hydroxymethyl)aminomethane (Tris, 99.9%) were purchased from J&K CHEMICA Co. Ltd. (Beijing, China). Butyl methacrylate (BMA, AR grade) was ordered from Bodi Chemical Reagent (Tianjin, China). Choline chloride (ChCl, AR grade), ethylene glycol (EG, AR grade) and α-methacrylic acid (MAA, AR grade) were supplied by Kemiou Chemical Co. Ltd. (Tianjin, China). Ethylene glycol dimethacrylate (EDMA, 98%) was from Sigma-Aldrich (St. Louis, MO, USA). Toluene was purchased from Tianjin Chemical Reagent Co. Ltd. (Tianjin, China). 1-Butyl-3-methylimidazolium

tetrafluoroborate

([BMIM]BF4,

98%),

and

1-hexyl-3-methylimidazolium tetrafluoroborate ([HMIM]BF4, 98%) were from Shanghai

Chengjie

Chemical

Reagent

(Shanghai,

China).

Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%) was obtained from

7

Aiwang Chemical Technology Co., Ltd. (Shanghai, China). Ammonium persulfate (APS) was from Shanghai Machlin Biochemical Co., Ltd. (Shanghai, China). N,N’-Methylene-bis-acrylamide (MBA, 99%), bovine hemoglobin (BHb), myoglobin (MyO) and cytochrome C (CytC) were supplied from Beijing Dingguochangsheng Biotechnology Co. Ltd. (Beijing, China). Dithiothreitol (DTT, 99%), bovine serum albumin (BSA, 96%), iodoacetamide (IAA, 98%), and trypsin (potency ≥ 2500 units/mg) were from Aladdin (Shanghai, China). Trifluoroacetic acid (99.0%) was obtained from Shanghai Machlin Biochemical CO., Ltd. (Shanghai, China). Bare fused-silica capillaries (100 μm ID, 375 μm OD) were supplied by Xinnuo Optic Fiber Plant (Hebei, China). 2.2 Instrumentation Capillary electrophoresis (CE) experiments were carried on CE7100 system (Agilent Technologies, America) with UV detector. A syringe pump (RSP04-B, RISTRON, Zhejiang) was used for protein injection. The FT-IR spectroscopy was measured by instrument Fourier transform infrared spectrometer (NICOLET380, Thermo Fisher Scientific, USA). N2 adsorption experiments was conducted on Aperture distributor (v-sorb 2800TP, Beijing jinepo technology co., LTD., Beijing). The performance of analytes were measured by Q-Exactive mass spectrometer (Thermo Fisher Scientific). The analytical column was C18 column (50 μm inner-diameter×15 cm, 2 μm C18). 2.3 Preparation of DESs The DESs used in this study consists of ChCl and EG. The reagents are dried

8

before use. ChCl was mixed with EG (1:3, mol/mol), and then placed at 100 ℃ oil bath for 2 h. The final prepared DESs was clear and homogeneous. 2.4 Preparation of monolithic capillary columns The fused-silica capillary was flushed with 1 mol/L NaOH followed by water for 30 min. Then 10% of γ-MPS in toluene solution was pumped through the capillary for 3 h. After rinsed with water, the capillary was dried with nitrogen gas. AIBN, BMA, MAA and EDMA were dissolved in a mixture of RTIL and DESs (Table 1). After 15 minutes of ultrasound, the prepolymerization solution was injected into the pretreated capillary of 50 cm with a syringe. With both ends sealed with rubbers, the capillary was placed in 53℃ water bath for 90 min. The lower and upper limits of monolith length of capillary was 5 and 40 cm, respectively, by control the length of the capillary immersed in the water bath. When polymerization was completed, acetonitrile was pumped through the capillary to remove the porogen and other soluble substances. Considering the rigidity and permeability of the resulting monolith, C1, C7 and C18 were selected for the immobilization of trypsin. 2.5 Immobilization of trypsin Trypsin solution and tris(2-carbonylethyl)phosphonate hydrochloride solution (1 mg/mL) were prepared in 20 mM Tris-HCl buffer (pH 8.0), and mixed with the volume ratio of 1: 10. The mixture was placed in water bath at 25°C for reaction of 3 h, and then centrifuged at 4°C for 10 min (10000 rpm) to remove insolubles. Then ammonium persulfate (1 mg/mL) and N,N'-methylenebisacrylamide (2 mg/mL) was added into the supernatant, respectively, to form the solution of enzyme derivative.

9

The enzyme derivative was injected into the capillary monolith synthesized and reacted at 25°C for 5 h. After completion of the reaction, the capillary was washed with 20 mM Tris-HCl buffer (pH 8.0) for 1 h to remove unreacted substances. 2.6 Enzymatic activity of immobilized trypsin The activity of the immobilized trypsin was evaluated by measuring the hydrolysis products of BAEE at different concentration. To reduce analysis time, the microreactors were cut into small fragments of 2 cm length. BAEE was dissolved in 20 mM Tris-HCl (pH 8.0), and pumped through the immobilized enzyme reactors at volumetric flow rate of 0.8 μL/min at 25℃. The peak area of hydrolysis products were analyzed by CE at 214 nm using a 20 mM Tris-HCl of pH 8.0 as running buffer. 2.7 Michaelis constant and maximum reaction rate determined by capillary electrophoresis In this experiment, the capillary used was 50 cm long with 5 cm of microreactor. The capillary end was 8.5 cm away from the detection window, and the effective length was 41.5 cm. 20 mM Tris-HCl (pH 8.0) was used as the running buffer. The substrate was 5 mM BAEE and prepared in the running buffer. The enzyme reactor was first equilibrated with the separation buffer for 10 min before injection. The substrate was injected on the enzyme reactor for a certain period of time. The separation voltage was 15 kV and the detection wavelength was 214 nm. 2.8 Proteins sample preparation The liver of rat was cleaned with 0.9% NaCl to remove blood and other contaminants, then cut into small pieces and homogenized in 50 mM PBS (pH 7.4)

10

containing 2 mol/L NaCl and 1% (v/v) protease inhibitor at an ice bath. The resulting complex cocktail was centrifuged for 1 h at 1600×g. Bradford assay was used to measure the concentration of protein. BSA, CytC, BHb, MyO, and the proteins extracted from rat liver were dissolved respectively, in 50 mM Tris-HCl (pH 8.0) containing 8 mol/L urea and reduced in 100 mmol/L DTT for 20 min in 50℃. After cooled to room temperature, the samples were alkylated in 100 mmol/L IAA for 20 min in the dark, followed by the dilution with 50 mmol/L Tris-HCl (pH 8.0). For a comparison, a digestion in solution was conducted by mixing trypsin with pretreated protein at a mass ratio of 1:50 (w/w), then the solution was placed in 37 °C water bath for 12 h. 2.9 HPLC-MS/MS experiments The peptides from either off-digestion based on the monolithic IMER or in-solution digestion were desalted before HPLC-MS/MS analysis to prevent the non-volatile salt above from damaging LC-MS instrument. In brief, C18 ziptip commercial column (Merck Millipore Ltd, Tullagreen, Carrigtwohill, Co. Cork, IRL) was used to remove salt in the IMER according to the conventional procedure in proteomics. Then the sample was injected into a Nano-LC system (EASY-nLC 1000, Thermo Fisher Scientific). The analytes were separated by C18 column at the flow rate of 300 nL/min. The phase A of the mobile phase was an aqueous solution containing 0.1% trifluoroacetic acid, and the phase B was 0.1% trifluoroacetic acid in acetonitrile. The HPLC elution gradient was follows: 1) 0-60 min, 5%-35% B; 60-65

11

min, 35%-90% B; 65-75 min, 95% B. The source was operated at 2.4 kV. The mass spectrometric analysis was carried out in a data-dependent mode. For MS1 survey scan, the target of automatic gain control was 1e6 and the resolution was 70,000. The MS2 spectra were obtained at a resolution of 17,500. 2.10 Database searching Proteome Discoverer software was used for searching MS/MS data with an overall false discovery rate (FDR) for peptides of less than 1%. Proteins with a score<2 were removed. Trypsin specificity was evaluated by the search of peptide sequences with a maximum of two missed cleavages. Carbamidomethylation on cysteine was designated as fixed modification. Oxidation of acetylation and methionine on protein N-terminal were set as variable modifications. Mass tolerances were set at ±10 ppm for precursor ions and ±0.02 Da for MS/MS. 3. Results and discussion 3.1 Preparation of monolithic IMER Monolithic materials are usually used for enzyme immobilization supports since the surface of the monolith can be well modified. In this case, the monolithic supports were prepared by monomers MAA, BMA, and EDMA. MAA was added in the polymerization

for

obtaining

more

hydrophilic

support

suitable

for

the

immobilization of trypsin to digest higher-molecular weight proteins, BSA. To obtain monolithic columns with desired porous properties, the conditions of monoliths synthesis based on the MAA-BMA-EDMA copolymer were optimized (Table 1). The mixture of DESs composed of ChCl and EG and [BMIM]BF4 or [HMIM]BF4 were

12

chosen as the optimal porogen. The double bonding of the monolithic column required for click reaction can be demonstrated by infrared spectroscopy (Fig. 2), in which a significant C=C stretching vibration peak was observed at 1638 cm-1, 2960 cm-1 was the CH3 stretching vibration, 1166 cm-1 was the C-O stretching vibration and 1723 cm-1 was a typical C=O stretching vibration. The results meant the presence of a large amount of double bond in the formulation provided. According to the thiol-ene click reaction, trypsin was immobilized via the double bonding of the surface of the monolith. It was found that the amount of BMA had affected the performance of monolithic matrix and the subsequent enzymatic hydrolysis. Reducing the amount of BMA had a significant negative effect on the enzymatic hydrolysis of the resulting IMER, which may be attributed to higher hydrophobic of the resulting monolith due to increased percentage of EDMA in the pre-polymerization mixture. We also investigated the influence of the type of RTIL on the monolithic matrix thus enzyme reactor. When [BMIM]BF4 was used as the porogen, the enzymatic hydrolysis of the enzyme reactor was not ideal. It may be that the carbon chain length of the cationic part in the RTIL affected the morphology of the monolithic column and structure, which in turn influenced the fixed amount of trypsin (Fig. S1). 3.2 Characterization of monolith The internal morphology of the monoliths and in proximity of the capillary wall (Fig. 3a) was investigated using scanning electron microscopy (SEM). Fig. 3b and 3c show the distinct cauliflower-like structure of the monolith. In addition, the

13

monolithic support is homogeneous and tightly attached to the capillary inner wall. The morphology of the IMER after the enzyme immobilization on the monolith is shown in Fig. 3d, e and f. The macropores in the monolithic IMER allowed the enzymolysis at higher flow rate with low back pressure, leading to improved mass transfer and high enzymatic digestion rate. N2 adsorption experiments were used to investigate the pore properties of the monolith. The structure of the monolithic supports was composed of macropores and micropores with average pore width of 33.1 nm (Fig. 4a). BET surface area was 12.82 m2/g, which may offer enough surface for enzyme immobilization. All the polymers showed type IV isotherm [30], suggesting the feature of mesopores polymer (Fig. 4b). The type of H3 loops with no limit of adsorption at high p/p 0 was observed, suggesting a substantial amount of slit-like pores. In addition, the steepness of the desorption branch from the hysteresis loop of the monolith suggests that the throats connecting the cavities on these polymers are quite uniform in size. 3.3 Performance of monolithic IMERs Capillary electrophoresis (CE) was used to detect the in-line enzymatic hydrolysis of the enzyme reactor since it is a promising technique as high efficiency and sensitivity, low sample consumption, and fast analysis. The length of the IMER was 5 cm, and the substrate for enzymatic hydrolysis was 5 mM BAEE. Table S2 displays influence of pH from the background electrolyte (BGE) on separation. The higher the pH value was, the lower the retention factor of the analyte was except pH 10. Access to BGE pH values in the range 7-10 creates the possibility of manipulation

14

of separation selectivity based on the charge of the analytes that may change when varying the pH. The effect of BGE pH on resolution (Rs) between BAEE and BA and indicates that the protonation of BAEE at pH 7 resulted in slower migration of this species and greatly improved resolution. However, all of the pH brought good separation (R s > 1.5) in spite of maximum resolution obtained at pH of 9.0. Considering the enzymatic efficiency of trypsin and the effect of CE separation, 20 mM Tris-HCl buffer at pH 8.0 was selected as the enzymatic hydrolysis and subsequent electrophoresis conditions (Fig. 5a). Fig. S5 displays the effect of enzyme concentration on the performance enzymatic hydrolysis of the IMER. Different concentrations of trypsin (5, 10, 15 mmol/L) were pumped through the monolith for a constant time. The relative enzyme activity, in terms of peak area of BA, increased significantly with increasing trypsin concentrations from 5 to 10 mmol/L. There was no significant change of the relative enzyme activity when the trypsin concentration was further increased to 15 mmol/L. As a result, 10 mmol/L trypsin was selected for enzyme immobilization. We further investigated the relationship between the length of the enzyme reactor and the enzymatic hydrolysis capacity (Fig. 5b). The peak area of product, BA, was measured by CE with different concentrations of BEAA as substrate at a flow rate of 2 μL/min in the enzyme reactor. Three concentrations of BAEE solution at 1 mM, 3 mM, and 5 mM were prepared, and the enzyme reactor lengths were 1 cm, 3 cm, 5 cm, and 7 cm, respectively. The results indicated that the peak area of BA increased with the increase of the length of the enzyme reactor. At high flow rate, increasing the

15

length of the enzyme reactor can improve the efficiency of enzyme hydrolysis to some extent. To further evaluate the enzymatic hydrolysis rate of the enzyme reactor, we prepared BAEE and BHb as substrated for IMER on-line digest. The total length of capillary was 50 cm with 5 cm IMER. Substrate solutions were injected into the IMER at 50 mbar for 15 s, then the capillary was suspended in buffer for certain seconds. The electric field of 15 KV was used to separate the analytes. Figure 6a showed the electrophoresis results of BAEE enzymatic hydrolysis for 5 s, 15 s, and 30 s, respectively, suggesting that the substrate can completely be hydrolyzed within 30 s on the enzyme reactor. Compared with the offline enzymatic hydrolysis, the relative intensity of the peak was unchanged, which demonstrating the validity of the method. Figure 6b was the electropherogram of on-line trypsin digestion of 1 mg/mL BHb with 50 s incubation. It demonstrated the protein was efficiently hydrolyzed into polypeptides on the enzyme reactor. Using a solution of 0.1-5 mmol/L BAEE in 20 mmol/L Tris-HCl (pH 8) as a substrate through the enzyme reactor with a length of 2 cm and reacting with the enzyme reactor for a different time, the product BA and the undigested substrate BAEE were separated and analyzed by CE to determine the activity of the immobilized trypsin. The Michaelis constant (Km) and the maximum reaction velocity (Vmax) obtained were 2.1 mM and 0.028 μmol/min, respectively. The Km value of the enzyme reactor was quite similar to that of the free trypsin (1.4 mM), indicating quite small diffusionl imitation in the IMER.

16

3.4 Reproducibility of monolithic enzyme reactor We used an optimally formulated enzyme reactor for reproducibility evaluation. The experiment was carried out using Tris-HCl (pH 8, 20 mM) as a mobile phase. Using BAEE as a substrate, continuous injection through the enzyme reactor was performed, and the retention time (tR) of BAEE and BA, and the relative standard deviation (RSD) of the peak area for BA were used to characterize the intra-column and inter-column reproducibility of the enzyme counter (Table 2). The RSD values of tR for the product BA were between 1.2% (n = 5) and 3.4% (n = 5) for the inter-batch and intra-column RSD values, respectively. The RSD of the peak area of BA for run-to-run and batch-to-batch were 6.7% (n = 5) and 3.9% (n = 5), respectively, which was better than that for the schemes of conventional tryptic reaction (RSD > 12%, n > 3). The RSD values between the batches were smaller than those between the columns, which may be attributed to the activity of the enzyme at room temperature. Previous study has shown that trypsin solution almost completely lost enzyme activity when placed at 25 °C for 24 h [27]. Since the enzyme reactor digests the substrate at room temperature, the activity of the enzyme is affected obviously to some extent after repeated use. However, the stability of the enzyme reactor column was still demonstrated. To investigate the ruggedness of the IMERs, the IMERs were placed in 60 mM Tris-HCl to measured enzyme activity repeatedly (Fig. S4). At the beginning, trypsin activity decreased rapidly, above 80% of the initial activity at the 5th cycle. The activity then decreased slowly and stabilized at 80% of the initial activity. It was

17

found that the enzyme activity remained ca. 75% of the initial activity after 20 times. The results indicated good ruggedness of the IMERs. 3.5 Protein digestion To demonstrate the feasibility of protein digestion using the thiol-ene-based IMERs, a comparison of ESI–MS analysis of the eluent from the IMER column (2 min) with that from free trypsin digestion (with incubation time of 12 h) was conducted. BSA was selected as a standard protein to test the digestion performance of the enzyme reactor with length of 5 cm under the condition of 37 ℃ at a flow rate of 0.5 μL/min. Table 3 shows that BSA can be well digested by both methods, and the number of identified peptides is 73 for conventional solution and 78 for enzyme reactor, respectively, and the coverage rates are 82.7% for conventional solution and 78.9% for enzyme reactor, respectively. Compared with the traditional method, the enzyme reactor can produce more peptides after enzymatic hydrolysis, while greatly shortened the time of enzymatic hydrolysis and possessed higher enzymatic efficiency. Further, a mixture of four proteins, consisted of CytC, MyO, BHb and BSA, was passed through the enzyme reactor. The results of the analysis were shown in Table 4. BHb is a tetrameric protein composed of two alpha chains and two beta chains, and the coverage of alpha and beta for BHb measured was 91.55% and 80.69%, respectively. The coverage of Cyt-C was 77.14%, and the coverage of MyO was 85.06%. However, the coverage of BSA in this mixture was only 36.08%. The decrease in the coverage obtained with the protein of larger molecular weight may be

18

attributed to the decreased concentration of the protein. For pure BSA solution, the concentration was 1 mg/mL, while the BSA concentration in the mixture of the four proteins was 0.25 mg/mL. After enzymatic hydrolysis, the concentration of the low abundance polypeptide was lower than before, the coverage rate reduced distinctly due to the limited detection of the instrument. This decrease in the coverage due to reduction of protein has been frequently observed, which was in consistent with the findings of Ma et al. [8]. 3.6 Analysis of proteins extracted from rat liver In order to investigate the application of the enzyme reactor in actual samples, the protein in rat liver was extracted and injected onto the enzyme reactor. The results of liver proteolysis were specifically described in Table S3. A total of 1034 proteins was identified, in which 19 of the proteins had the MW less than 10 kDa with the smallest MW of 4.1 kDa. In addition, 68 of the proteins had the MW greater than 100 kDa with the maximum MW of 509.4 kDa (Fig. 7a). The number of proteins with similar pI values was close, with 56 proteins between the pI value of 3-5 and 65 proteins greater than pI 10 (Fig. 7b). The above results demonstrated the capacity for the digestion of different proteins using the enzyme reactor was efficient and the potential of the enzymatic hydrolysis for actual sample. PANTHER

(Protein

Analysis

Through

Evolutionary

Relationships,

http://www.pantherdb.org/) is a database to analysis protein according to evolutionary relationships and functions. We used PANTHER to functionally categorize the identified proteins. The proteins were converted to their EntrezGene IDs and divided

19

into 21 categories according to their various application (Fig. 7c). The results indicated that the majority function of identified proteins in rat liver were oxidoreductase, nucleic acid binding, hydrolase and transferase. Gene ontology (GO) analysis was conducted using DAVID (the Database for Annotation, Visualization and Integrated Discovery, https://david.ncifcrf.gov/), in which bioinformatics resources, uniprot accession numbers of identified proteins were subjected to analysis. GO classification analysis showed that the proteins in different cellular components can be effectively extracted and identified (Fig. 7d). Figure 7d demonstrated the GO categories under cellular component include membrane 25.3%, cytoplasm 39.6%, cytoskelen 2.5%, nucleolus 5.8% and nucleus 30.7%. There have been a number of studies on enzyme-recombinant proteins in enzyme reactors [31-33]. In Table 5, we compared the enzymatic hydrolysis effects of rat livers with other reports. Although the number of proteins identified after enzymatic hydrolysis of the liver was lower than that of Wang et al. [32], our preparation method was simpler. In addition, one single trypsin enzyme was used in the click-based IMER compared to the dual enzyme reactor of Ref. 32. 4. Conclusions We prepared a monolithic enzymatic reactor based on click chemistry using green solvents DESs and RTILs as porogen. The poly(BMA-MAA-EDMA) monolith can be used as an excellent support to immobilize proteolytic enzymes with high activity for digestion of high-molecular weight proteins exceeding 150 000 Da. A rapid digestion of proteins extracted from rat liver was achieved using the new IMER

20

with 1034 proteins identified. Our immobilized enzyme reactor displays the advantages of a lower reaction temperature, shorter reaction time, as well as even a better peptide coverage. Although only immobilization of trypsin was demonstrated in this report, the method to immobilize other enzymes may also be feasible in view of the principle of the approach. In addition, the results for proteolysis digests in rat liver demonstrated the great potential of the prepared IMERs for proteome analysis with higher confidence.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments This work was supported by National Natural Science Foundation of China (Grant No. 21775109).

21

References [1] A. R. Joyce, B. Ø. Palsson, The model organism as a system: integrating 'omics' data sets, Nat. Rev. Mol. Cell Biol. 7 (2006) 198-210. [2] T. E. F. Walton, J. N., Rajan, Omics and anaesthesia: pharmacogenomics, proteomics and metabolomics, Anaesth. Intens. Care Med. 20 (2016) 248-253. [3] M. Getie-Kebtie, P. Franke, R. Aksamit, M. A. Alterman, Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach, J. Proteome Res. 7 (2008) 3697-3707. [4] M. X. Gao, P. Zhang, G. F. Hong, X. Guan, G. Yan, C. Deng, X. Zhang, Novel monolithic enzymatic microreactor based on single-enzyme nanoparticles for highly efficient proteolysis and its application in multidimensional liquid chromatography, J. Chromatogr. A 1216 (2009) 7472-7477. [5] Y. Zhang, B. R. Fonslow, B. Shan, M.-C. Baek, J. R. Yates III, Protein analysis by shotgun/bottom-up proteomics, Chem. Rev. 113 (2013) 2343-2394. [6] L.

N.

Amankwa,

W.

G.

Kuhr,

Trypsin-modified-fused-silica capillary

microreactor for peptide mapping by capillary zone electrophoresis, Anal. Chem. 64 (1992) 1610-1613. [7] E. Calleri, C. Temporini, F. Gasparrini, P. Simone, C. Villani, A. Ciogli, G. Massolini, Immobilized trypsin on epoxy organic monoliths with modulated hydrophilicity: Novel bioreactors useful for protein analysis by liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A 1218 (2011) 8937-8945.

22

[8] J. Ma, Z. Liang, X. Qiao, Q. Deng, D. Tao, L. Zhang, Y. Zhang, Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity, Anal. Chem. 80 (2008) 2949-2956. [9] X. Hu, J. Yang, C. Chen, H. Khan, Y. Guo, L. Yang, Capillary electrophoresis-integrated immobilized enzyme microreactor utilizing single-step in-situ penicillinase-mediated alginate hydrogelation: Application for enzyme assays of penicillinase, Talanta 189 (2018) 377-382. [10] M. Safdar, J. Spross, J. Jänis, Microscale immobilized enzyme reactors in proteomics: latest developments, J. Chromatogr. A 1324 (2014) 1-10. [11] Z. Xiao, L. Wang, Y. Liu, H. Khan, Y. Guo, L. Yang, A “plug-and-use” approach towards facile fabrication of capillary columns for high performance nanoflow liquid chromatography, J. Chromatogr. A 1325 (2014) 109-114. [12] L. Liu, B. Zhang, Q. Zhang, Y. Shi, L. Guo, L. Yang, Capillary electrophoresis-based immobilized enzyme reactor using particle-packing technique, J. Chromatogr. A 1352 (2014) 80-86. [13] N. Wu, S. Wang, Y. Yang, J. Song, P. Su, Y. Yang, DNA-directed trypsin immobilization on a polyamidoamine dendrimer-modified capillary to form a renewable immobilized enzyme microreactor, Inter. J. Biol. Macro. 113 (2018) 38-44. [14] Z. Yin, W. Zhao, M. Tian, Q. Zhang, L. Guo, L. Yang, A capillary electrophoresis-based immobilized enzyme reactor using graphene oxide as a support via layer by layer electrostatic assembly, Analyst 139 (2014) 1973-1979.

23

[15] L. D’Ulivo, J. Witos, K. Öörni, P. T. Kovanen, Marja.-L. Riekkola, Open tubular capillary electrochromatography: A useful microreactor for collagen I glycation and interaction studies with low-density lipoprotein particles, Anal. Chim. Acta 664 (2010) 185-189. [16] Q. Q. Li, F. Q. Yang, Y. Z. Wang, Z. Y. Wu, Z. N. Xia, H. Chen, Evaluation of thrombin inhibitory activity of catechins by online capillary electrophoresis-based immobilized enzyme microreactor and molecular docking, Talanta 185 (2018) 16-22. [17] J. W. Cooper, J. Chen, Y. Li, C. S. Lee, Membrane-based nanoscale proteolytic reactor enabling protein digestion, peptide separation, and protein identification using mass spectrometry, Anal. Chem. 75 (2003) 1067-1074. [18] H. Yuan, L. Zhang, Y. Zhang, Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid–silica hybrid monolith as matrix for on-line protein digestion, J. Chromatogr. A 1371 (2014) 48-57. [19] M. Kato, K. Inuzuka, K. Sakai-Kato, T. Toyo’oka, Monolithic bioreactor immobilizing trypsin for high-throughput analysis, Anal. Chem. 77 (2005) 1813-1818. [20] A. G. Pereira-Medrano, S. Forster, G. J. S. Fowler, S. L. McArthur, P. C. Wright, Rapid fabrication of glass/PDMS hybrid mIMER for high throughput membrane proteomics, Lab. Chip 10 (2010) 3397-3406. [21] P. L. Golas, K. Matyjaszewski, Marrying click chemistry with polymerization: expanding the scope of polymeric materials, Chem. Soc. Rev. 39 (2010)

24

1338-1354. [22] M. Vodopivec, A. Podgornik, M. Berovic, A. Strancar, Characterization of CIM monoliths as enzyme reactors, J. Chromatogr. B 795 (2003) 105-113. [23] M. Wu, H. Zhang, Z. Wang, S. Shen, X. Chris Le, X.-F. Li, “One-pot” fabrication of clickable monoliths for enzyme reactors, Chem. Commun. 49 (2013) 1407-1409. [24] C. E. Hoyle, C. N. Bowman, Thiol–ene click chemistry, Angew. Chem. Int. Eng. 49 (2010) 1540-1573. [25] Y. Chen, M. Wu, K. Wang, B. Chen, S. Yao, H. Zou, L. Nie, Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene “click” strategy, J. Chromatogr. A 1218 (2011) 7982-7988. [26] T. Welton, Room-temperature ionic liquids. Solvents for synthesis and catalysis, Chem. Rev. 99 (1999) 2071-2084. [27] A. P. Abbott, D. Boothby, G. Capper, D. L. Davies, R. K. Rasheed, Deep eutectic solvents formed between choline chloride and carboxylic acids:   Versatile alternatives to ionic liquids, J. Am. Chem. Soc. 126 (2004) 9142-9147 [28] G. Li, W. Wang, Q. Wang, T. Zhu, Deep eutectic solvents modified molecular imprinted polymers for optimized purification of chlorogenic acid from honeysuckle, J. Chromatogr. Sci. 54 (2016) 271-279. [29] L. S. Zhang, S. P. Gao, Y. P. Huang, Z. S. Liu, Green synthesis of polymer monoliths incorporated with carbon nanotubes in room temperature ionic liquid

25

and deep eutectic solvents, Talanta 154 (2016) 335-340. [30] K.S.W. Sing. Reporting physisorption data for gas/solid systems with special reference to the determination of surface area and porosity, Pure Appl. Chem. 54 (1982) 2201-2218. [31] J. Ma, C. Hou, Y. Liang, T. Wang, Z. Liang, L. Zhang, Y. Zhang, Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith, Proteomics 11 (2011) 991-995. [32] B. Wang, L. Shangguan, S. Wang, L. Zhang, W. Zhang, F. Liu, Preparation and application of immobilized enzymatic reactors for consecutive digestion with two enzymes, J. Chromatogr. A 1477 (2016) 22-29. [33] Z. Zhang, L. Zhang, C. Zhang, W. Zhang, Hybrid organic-inorganic monolithic enzymatic reactor with SBA-15 nanoparticles incorporated, Talanta 119 (2014) 485-491.

26

Legends

Fig. 1 Scheme of the monolithic microreactor.

Fig. 2 FT-IR spectra of monolith of poly(MAA-co-BMA-co-EDMA).

Fig. 3 SEM images of monolith in capillaries (a, d×1400) and microchannels (b, e×10000, c, f×20000)

Fig. 4 The pore size distribution (a) and nitrogen sorption isotherms of the monolithic support (b). 27

Fig. 5 Effect of the pH of Tris-HCl on IMER for hydrolyzing BAEE (a) and the electropherograms of different length of IMER hydrolyzing BAEE (b). 5 mmol/L BAEE solution was injected into 3 cm length of IMER at the flow rate of 0.5 μL/min. UV detection was carried out at 214 nm. The CE buffer was 20 mM Tris-HCl.

Fig. 6 Electropherograms of IMER hydrolysis of 5 mM BAEE at different incubation times (a) and the electropherogram of 1 mg/mL BHb digested on-line with 50 s incubation (b). The CEC condition was same as Fig. 5.

28

Fig. 7 Molecular weight (a), isoelectric point distribution (b), protein classification (c) and Gene Ontology annotations for cellular component (d) of proteins in rat liver identified by the database.

29

Table 1 Composition of monoliths AIBN

MAA

BMA

EDMA

(mmol)

(mmol)

(mmol)

(mmol)

(μL)

(μL)

C1

1.0

0.096

0.156

0.072

295

130

C2

1.0

0.096

0.288

0.096

194

130

C3

1.0

0.096

0.312

0.072

194

130

C4

1.0

0.096

0.326

0.058

194

130

C5

1.0

0.096

0.341

0.043

194

130

C6

1.0

0.096

0.312

0.072

295a

130

C7

1.0

0.096

0.312

0.072

295b

130

C8

1.0

0.096

0.312

0.072

260

130

C9

1.0

0.096

0.312

0.072

156

130

C10

1.0

0.096

0.312

0.072

194

130c

C11

1.0

0.096

0.312

0.072

194

130d

C12

1.0

0.096

0.312

0.072

194

130e

C13

1.0

0.096

0.312

0.072

194

130f

C14

1.0

0.096

0.312

0.072

194

130g

C15

1.0

0.096

0.312

0.072

194

130h

C16

1.0

0.096

0.312

0.072

194

130i

C17

1.0

0.096

0.312

0.072

194

130j

C18

1.0

0.096

0.312

0.072

295

80

Column

a, b

[HMIM]BF4 ChCl/alcohols

The ionic liquid was [OMIM]BF4, [BMIM]BF4, respectively.

c, d, e, f, g, h

The alcohol of DESs was methanol, ethanol, n-propyl alcohol, n-butyl

alcohol, respectively. i, j

The ratio of choline chloride to ethanediol was 1:2, 1:4, respectively.

30

Table 2 Relative standard deviation (RSD) of reproducibility on IMER. Run-to-run,%

Batch-to-batch,%

tR of BA (n = 5)

1.2

3.4

Area of BA (n =5 )

6.7

3.9

Table 3 Polypeptide identified of the trypsin digestion of BSA using the IMER and the free trypsin. Digestion

Sequence

Unique

coverage (%)

peptides

Accession No. methods In-solution

P02769

82.7%

73

Microreactor

P02769

78.91%

78

Table 4 Database searching results of a four-protein mixture digests by IMER Sequence Protein

Unique

Accession

MW (kDa) coverage (%)

peptides

P01966

91.55

12

15.2

P02070

80.69

18

15.9

BSA

P02769

36.08

38

69.2

Cyt-C

P00004

77.14

15

11.8

MyO

P68082

85.06

18

17.1

BHb subunit α BHb subunit β

31

Table 5 An overview on IMER for rat liver proteolysis digests Material

Numbers of proteins

GLYMO-IDA-silane Hybrid monolith with SBA-15 particles Hybrid monolith with SBA-15 particles Poly(butyl methacrylate-co-α-methacrylic acid-co-ethylene glycol dimethacrylate)

541

Flow rate (μL/min) 0.3

1091

0.5

[32]

11

0.5

[33]

1034

0.5

Present work

32

Ref. [31]