Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin

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Journal of Chromatography A, xxx (2016) xxx–xxx

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Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin a,b ´ Kinga Meller a , Paweł Pomastowski a,b , Damian Grzywinski , Michał Szumski a,b,∗ , a,b Bogusław Buszewski a b

Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toru´ n, Gagarina 7, 87-100 Toru´ n, Poland Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University in Toru´ n, Wile´ nska 4, 87-100 Toru´ n, Poland

a r t i c l e

i n f o

Article history: Received 10 November 2015 Received in revised form 22 January 2016 Accepted 23 February 2016 Available online xxx Keywords: Trypsin Chymotrypsin Transferrin Immobilized enzyme microreactor Proteomics Capillary liquid chromatography

a b s t r a c t The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (␥-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (␮-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (N␣-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Enzymatic reactions, due to their high specificity and selectivity, constitute a very attractive solution in many branches of industry as well as a valuable analytical tool [1,2]. On the laboratory scale, the preparation of microfluidic reactors with enzymes immobilized on the solid, monolithic support is one of the ways of using enzymes [3,4]. Such an approach combines the advantages of miniaturization, monolithic beds as a supports, and enzymes as environmentally friendly, specific, and selective biocatalysts. Additionally, the microfluidic ␮-IMER may be easily connected to a subsequent capillary system which enables its coupling with separation and detection techniques. Such a system prevents the reaction products from being contaminated or lost. With regard to the application and the type of an immobilized biocatalyst (e.g. glu-

∗ Corresponding author at: Chair of Environmental Chemistry and Bioanalytics, ´ Gagarina 7, 87-100 Faculty of Chemistry, Nicolaus Copernicus University in Torun, ´ Poland. Fax: +48 566114837. Torun, E-mail address: [email protected] (M. Szumski).

cose oxidase [5], lipase [6], acetylcholinesterase [7], l-asparaginase [8]), the microfluidic reactors enable carrying out highly specific reactions with minimal manual handling, reagent consumption, and time-effectively. ␮-IMERs can be particularly useful in proteomic research, e.g. for the identification of proteins with PMF technique (Peptide Mass Fingerprinting). The PMF is based on the initial digestion of a protein of interest by a specific enzyme and a subsequent MS analysis of the resulting polypeptides. The pattern of peaks recorded on the MS spectrum is finally compared with the theoretical masses available in proteomic databases [9]. Trypsin is the most common of the proteolytic enzymes immobilized in microfluidic monolithic reactors. It is also widely discussed in available literature [10–16]. However, it is possible to immobilize some other proteases for protein digestion e.g. chymotrypsin [17,18], papain [19–21], or pepsin [22,23]. It is important to notice that the application of proteolytic enzymes for protein digestion is not limited only to the qualitative analysis in proteomic research involving the use of one, highly specific enzyme. It may also be employed in protein quantification and the investigation of the post-translational modifications, e.g.

http://dx.doi.org/10.1016/j.chroma.2016.02.070 0021-9673/© 2016 Elsevier B.V. All rights reserved.

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glycosylation [24]. In both cases, the fragmentation of the proteins to low-molecular weight polypeptides is the main aim of the proteolysis. Then, the quantitative analysis of the target protein may be performed on the basis of one specific peptide formed during enzymatic digestion, e.g. by AQUA analysis, with the use of labeled internal standards [25]. Such a fragment, selected as a representative, cannot occur in other polypeptides. The situation may be caused by missed cleavage sites. It is directly related to the efficiency of proteolysis. On the other hand, for research focused on the protein glycosylation, it is important to obtain short glycopeptide chains. Taking into account the fact that the digestion with trypsin usually leads to obtaining long glycopeptides with several glycosylation sites, it is more reasonable to use the enzyme which acts on more amino acid residues or to apply a mixture of nonspecific endoproteases for glycosylated protein digestion. For such a purpose, the preparation of a pronase-immobilized reactor was justified [26]. Tryptic digestion is also limited in the case of Laser-Induced Fluorescence (LIF) detection due to the slow reaction between a fluorescent reagent and resulting polypeptides, and, on the other hand, because the derivatization of an intact protein limits the trypsin activity to an arginine residue “cleave sites”. Such a situation, in turn, leads to obtaining a small number of fragments. The above mentioned problems justify the immobilization of chymotrypsin for digestion of protein in the case of the microreactor coupling with CE-LIF [27]. The same issue is also important in the case of MS detection because tryptic digestion of a protein containing a small number of arginine and lysine residues may lead to obtaining a few fragments which are too long for the MS analysis. In such a case, the proteins can be digested by several proteases of different activities to improve the protein fragmentation and to obtain a greater number of fully sequenced peptides. Such an approach can result in the higher sequence coverage and is particularly important when the reliable identification of one target protein is needed. The creation of an online system composed of two coupled microreactors (chymotrypsin- and trypsin-based) is one of the ways to avoid classical tandem reactions performed in solutions with dissolved enzymes [28]. Such a system allowed obtaining higher sequence coverage for ␤-casein (70%) when compared to single-enzyme reactors with immobilized trypsin (14%) or chymotrypsin (57%) in reaction time of 20.8 min. In a comparison, the classical digestions performed in solutions for 18 h and employing these enzymes resulted in the sequence coverage of 21% and 45% for trypsin and chymotrypsin, respectively. Recently, for protein digestion, a dual-microreactor with coimmobilized trypsin and chymotrypsin has been prepared with the use of a monolithic support on which trypsin was bound and nanoparticles used as a support for chymotrypsin [29]. In this case, a sample of proteins isolated from the rat liver were digested with dual-enzymatic system which allowed for the identification of 620 individual proteins while the total number of proteins identified with two single protease digestions was 606. The immobilization of the enzymes on the polymeric support containing glycidyl methacrylate (GMA) as a functional monomer may be performed by the aminolysis of the epoxy groups and then activation with dialdehyde. Research data [30,31] confirmed the fact that the greater the distance between the support surface and immobilized enzyme molecules is created, the higher efficiency of enzymatic reactions is achieved. It justifies the immobilization of trypsin onto membranes [32] or magnetic nanoparticles [33] functionalized with hexanediamine. It was also proved that papain immobilized on the GMA-co-EDMA monolith through hexanediamine chain exhibits activity higher than in the case of a reaction involving hydrolysis of an epoxy ring and oxidation with NaIO4 (despite higher immobilization capacity in the second case) [19]. This is probably due to the fact that the spacer arm prevents the enzyme from changing its conformation (by minimized interac-

tion between the support surface and biomolecules) and reduces the steric hindrances, thereby enhancing the formation of enzymesubstrate complexes. For this reason, such a technique was used in the present research. In contrast to the previously mentioned approach [29], the simultaneous immobilization of both trypsin and chymotrypsin reduces the preparation stages and amount of reagents required, i.e. one type of material is used as a support and one enzymatic preparation is used during immobilization, which is more preferable from economical and practical points of view. Similar methodology was used during the modification of a classical size monolithic silica column modified with epoxy groups (100 mm × 4.6 mm i.d.) by directly bonding two enzymes (without the spacer arm) using, however, two individual separate enzyme preparations [34]. Due to the fact that enzymatic reagents containing one particular enzyme of high purity are expensive, the verification of low-cost reagents with regard to their potential applications is economically favorable. Taking into account all the above-mentioned factors and numerous applications of proteolytic digestion, the main aim of this research was to prepare and evaluate a microfluidic reactor with two enzymes immobilized on a monolithic GMA-co-EDMA support using a low-cost, commercially available reagent (containing trypsin and chymotrypsin). The first stage of our research involved the selection of appropriate polymerization conditions (including porogen solvent composition and polymerization temperature) to obtain the support of good permeability, homogenous structure, and relatively high surface area. The activity of immobilized trypsin was evaluated with its commercially available substrate (BAEE) which enables the establishment of the optimal conditions for the hydrolysis reaction to be established. The activities of the both immobilized enzymes were tested by the digestion of transferrin which is known to be a protein relatively resistant to trypsin [35,36]. Therefore, the digestion in a dual-enzyme microreactor was compared with a classical digestion method (as a reference) in which a high-purity trypsin was used. The eluate from the microreactor and the post-reaction mixture were analyzed with MALDI-TOF MS.

2. Material and methods 2.1. Materials Fused-silica capillaries (150 ␮m i.d. × 375 ␮m o.d.) were purchased from, CM Scientific Ltd. (Silsden, United Kingdom), 3-(trimethoxysilyl) propyl methacrylate (␥-MAPS), glycidyl methacrylate (GMA), ethylene dimethacrylate (EDMA), 1dodecanol, cyclohexanol, 1,6-hexanediamine, glutaraldehyde, sodium cyanoborohydride, benzamidine, N␣-benzoyl-l-arginine ethyl ester (BAEE), Trypsin Gold, Mass Spectrometry Grade (used for transferrin digestion in a solution), human transferrin (used as a model protein), and trifluoroacetic acid (TFA) were purchased from Sigma–Adrich (Steinheim, Germany). The enzyme preparation used during the microreactors synthesis was obtained from Biological Industries (trypsin: 365.47 USP-u/mg, chymotrypsin: 151.23 USP-u/mg). The storage solution (containing sodium azide) used for flushing and storage of the microreactors was purchased from MACS Miltenyi Biotec (MEDianus Sp.z o.o., Kraków, Poland). Acetone, toluene, methanol, dichloromethane, sodium hydroxide, sodium bicarbonate, sodium phosphate monobasic dihydrate, ammonium bicarbonate (all of analytical grade), acetonitrile (HPLC ultra gradient grade) were purchased from Polskie Odczynniki Chemiczne (POCh, Gliwice, Poland). Deionized water was obtained from the Mili-Q ultrapure water producing system (Millipore, Bedford, MA, USA). Azobisisobutyronitrile (AIBN) and all the

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chemicals supplied for the MALDI-MS analyses were at the highest commercially available purity by Fluka Feinchemikalien (Neu-Ulm, Germany; a subsidiary of Sigma–Aldrich). Ground steel targets (Bruker Daltonics, Bremen, Germany) were used for sample deposition. ␣-Cyano-4-hydroxycinnamic acid and sinapic acid (SA) were employed as matrices for the MALDI analysis of tryptic digest and intact proteins, respectively (dried droplet method) [37]. Peptide Calibration Standard II and Protein Calibration Standards I and II, all from Bruker Daltonics, were used for external calibration. 2.2. Instrumentation A syringe pump NE-1002X (New Era Pump Systems, Farmingdale, NY) was used to pass the reagents (during the modification) and the substrate solution through the ␮-IMER. For the reactions carried out at constant temperature, the thermostat (Julabo, type F25) connected to a specially designed heat exchanger was used. A pneumatic HPLC pump (Knauer GmbH, Berlin, Germany) was used for columns flushing (after monolith synthesis) and for permeability measurements. All the chromatographic experiments were performed in a nanoLC system consisting of a 1260Capillary Pump (Agilent Technologies, Waldbronn, Germany), a 10-port nanoLC valve (model C72MX-6690D Vici-Valco, Schenkon, Switzerland), and a Crystal 100 UV–vis detector (Thermo Separation Products, San Jose, CA, USA). The system was controlled with the Clarity software (DataApex, Prague, Czech Republic). All the MS spectra were obtained with the MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a modified neodymium-doped yttrium aluminium garnet (Nd:YAG) laser (smartbeam II) operating at the wavelength of 355 nm and frequency of 2 kHz. 2.3. Capillary pretreatment and modification Initially, the capillary (150 ␮m i.d. × 375 ␮m o.d.) was flushed with acetone and dichloromethane and then dried by flushing with nitrogen. The modification of the inner surface of the fused silica capillary involved two steps: etching with 1 M NaOH solution (performed at 100 ◦ C for 3 h) and silanization with 10% solution of ␥-MAPS in toluene (performed at room temperature for 2 h). 2.4. The preparation and characterization of the monolithic support To obtain a monolithic bed suitable for further experiments, three polymerization temperatures (60, 70, 80 ◦ C) and several compositions of a porogen solution (with different cyclohexanol to 1-dodecanol ratios, herein referred to as weight percentage of 1dodecanol (%D )) were tested. In each case, the same composition of the monomers mixture (GMA/EDMA = 3/2 (w/w)) and the initiator content (1% AIBN (w/w) with respect to the total monomers weight) were used. The prepared polymerization mixtures were sonicated, vortexed, and purged with nitrogen (to eliminate oxygen), and then introduced into capillaries using a syringe. Both ends of the capillary were sealed with pieces of GC septa, and then, the capillary was placed in water bath. The polymerization was carried out at the temperature values of: 60, 70, and 80 ◦ C for 24 h. The permeability of the obtained monoliths (KF ) was evaluated on the basis of the measurement of acetonitrile flow rate through the capillaries at a given pressure set on the pneumatic LC pump. Then, permeability was calculated using the following formula (Eq. (1)): KF =

FL  2  m Pr 2

(1)

3

where −acetonitrile viscosity [Pa s], F − flow rate [␮l/min], L −the capillary length [dm], r −the capillary radius [dm], P − pressure [Pa]. The syntheses were performed in several capillaries (2, 3, or 4) which were subject to the same permeability tests (to verify the reproducibility of the obtained results) and then checked for homogeneity with a scanning electron microscope (SEM). The macroscopic samples of the monolithic supports of the highest permeability and homogeneous structure were analyzed using the nitrogen adsorption-desorption method to determine their specific surface area (SBET ). 2.5. Immobilization of the enzymes The aminolysis of epoxy rings was carried out using 10% 1,6-hexanediamine solution in a buffer at 80 ◦ C for 2.5 h. The activation with carbonyl groups was performed using a glutaraldehyde solution in a buffer of pH 8.5, at room temperature, for 3 h. The immobilization was carried out with a 3.5 mg/ml enzymatic reagent solution containing benzamidine (50 mM) at 4 ◦ C for 24 h. Finally, the imine bonds were reduced with NaCNBH3 , at room temperature, for 2 h. 2.6. Evaluation of -IMER activity and effect of selected factors on the yield of the reaction The activity of the prepared ␮-IMER was evaluated on the basis of trypsin-catalyzed hydrolysis of the N␣-benzoyl-l-arginine ethyl ester (BAEE) as a substrate. This compound is one of those most commonly used in the determination of trypsin activity (using UV–vis spectrometry [38], CE [16,39], LC [40,41] or MS [42]). Both of the reagents show similar UV–vis absorption spectra with one exception, i.e. at the wavelength of ␭ ≈ 253÷254 nm the product (carboxylic acid, N␣-benzoyl-l-arginine, BA) exhibits greater absorption than the substrate (ester, BAEE) [40,41]. Firstly, a series of standard solutions of the substrate (in 0.05 mM PBS, pH 8.0) in the concentration range of 0.5÷75 mM was directly injected into the separation column to find the BAEE peak and to prepare the calibration curve. Then, each of the substrate solutions was passed through the ␮-IMER at different flow rates: 0.25, 0.50, 0.75 and 1.00 ␮l/min (which corresponds to the linear velocity of 0.350, 0.617, 0.883 and 1.130 mm/s, respectively) using a syringe pump. Additionally, 25.0 mM BAEE solution was passed through the ␮-IMER at different temperature values: 15, 20, 30, and 37 ◦ C. In each case, the eluate from the ␮-IMER was continuously passing through the injection loop, and, at the proper moment, it was injected into the chromatographic column (Fig. 1). Accordingly, the reaction product, N␣-benzoyl-l-arginine (BA), was separated from the substrate, N␣-benzoyl-l-arginine ethyl ester (BAEE), and the degree of hydrolysis (%H) was calculated based on the peak area of the substrate from the difference between BAEE concentrations in the solutions before and after the reaction was carried out in the ␮-IMER. For calculations, the calibration curve and the following formula (Eq. (2)) were used: %H =

CBAEE,st − CBAEE × 100 [%] CBAEE,st

(2)

where CBAEE,st − standard BAEE solution concentration before passing through a ␮-IMER [mM], CBAEE −BAEE concentration in solution after passing the standard solution through a ␮-IMER [mM]. The separation was performed using a nanoLC system equipped with a homemade column packed with the octadecyl stationary phase. The mobile phase consisted of water and acetonitrile (both with the addition of TFA) at a flow rate of F = 2.0 ␮l/min. The chromatographic process initiated with 20% of the ACN content in the mobile phase, and then, the content was increased to the final 40%

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Fig. 1. (A) Diagram of chromatographic system used during calibration curve preparation and finding the peak derived from BAEE with examples of chromatograms obtained for three substrate concentrations, (B) diagram of chromatographic system with connected microreactor used for reaction reagents separation and finding the peak derived from BA with examples of chromatograms obtained after reaction.

value of ACN. Firstly, detection was performed at the wavelength of ␭ = 253 nm (according to the standard procedure), but finally, 30 nm lower wavelength was used (according to Ref. [41]). It was more preferable considering the fact that the reaction yield was evaluated on the basis of the substrate peak area (Eq. (2)) which, indeed, was almost three times greater at ␭ = 223 nm in comparison with the detection at ␭ = 253 nm. 2.7. Protein digestion and MALDI-TOF identification Transferrin was dissolved in a 50 mM ammonium bicarbonate (ABC) buffer of pH 8.05 to obtain the final protein concentration of 45 pm/␮l. The sample was passed through a ␮-IMER using a syringe pump at 37 ◦ C and flow rate of F = 0.05 ␮l/min. It corresponds to the residence time of 597 s ≈ 10 min (capillary of 8.0 cm in length). The eluate was collected in a plastic vial (for 60 min) and then subject to analysis using MALDI-TOF. 1 ␮l of the eluate was mixed with a matrix at the ratio of 1:1, and finally, 0.5 ␮l spots were deposited on the MALDI target [37]. In the case of the classical digestion method, the protein was dissolved in the same buffer (50 mM ABC, pH 8.05) and incubated with trypsin at the enzyme:substrate ratio of 1:50. The reaction was carried out for 14 h and terminated by the addition of 5 ␮l of 10% TFA. Mass spectra of transferrin hydrolysates were recorded in the positive mode using 25 kV acceleration voltage within a m/z range of 0–2500 Da (500 shots for one manual acquisition to the final of 2500). One-dimensional mass spectra were acquired and processed with the dedicated software, flexControl and flexAnalysis, respectively (both from Bruker Daltonik). The obtained MS peak lists were submitted to a Mascot search with SwissProt database for protein identification with the use of BioTools and ProteinScape software (both from Bruker Daltonik). Here, the following non-standard search parameters were used: cysteines modified with carbamidomethylation and mass tolerance set to 0.1 Da. Theoretical transferrin cleavage sites were determined using PeptideCutter, and the masses of fragments were calculated by PeptideMass (ExPasy Bioinformatics Resource Portal). Transferrin sequence was obtained from the UniParc database (UniProt). Intact transferrin mass spectrum was recorded in the linear-positive mode using 25 kV acceleration voltage within a m/z range of 10,000–80,000 Da and 200–1600 Da. To obtain the sequence coverage for the digested transferrin, the mass spectra recorded for the eluate from the microreactor were subject to anal-

Fig. 2. Effect of porogen solvent composition (%D means the percentage content of 1-dodecanol) on the monolith permeability.

ysis using SequenceEditor programme with the “TrypChymo” set as enzymes and the following parameters: partials: 2, error: 0.2 [Da], mass range 500.00÷16,000.00. 3. Results and discussion 3.1. Preparation of the monolithic support for immobilized enzymes Initially, a series of syntheses was performed at 60 ◦ C using different contents of 1-dodecanol in a porogen solvent (%D ): 0, 5, 10, 15, 20, 30, 40, 50, 60, 100 [%]. From among the obtained copolymers, the greatest permeability was presented by the monolith in which 15% of 1-dodecanol in the porogen solvent was used (KF = 1.71 × 10−14 m2 ). The results indicated that raising the content of a less polar component (1-dodecanol) to the amount above 15% led to the decrease in permeability (Fig. 2). This was probably due to the fact that growing polymer chains were more preferably solvated by individual monomers than by the porogen solvent which led to the increase in the size of the formed globules and decrease in the radius of the flow-through pores. Thus, 1-dodecanol at lower concentrations led to the increased permeability of the bed (due to the faster precipitation of nuclei and

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Fig. 3. SEM images of prepared monolihic beds (10,000×).

the formation of large pores between globules) whereas, at higher concentrations, it contributed to drastic swelling of the individual globules and decrease in the support permeability. It is noticeable on the images obtained from the scanning electron microscope (Fig. 3). In the cases of higher polymerization temperatures (70, 80 ◦ C) five different porogen solvent compositions were tested (0, 15, 30, 60, 100 (%D )). At higher temperatures, the polymerization initiator decomposed into radicals faster. It resulted in the increased initial amount of the polymerization nuclei which led to the smaller final size of the globules and a more homogenous structure of copolymers (in comparison with the beds synthesized at 60 ◦ C). Additionally, together with an increase in temperature (at the same composition of the porogen solvent), the precipitated nuclei were more soluble in the solvent and it probably led to the better permeability of the beds synthesized at 70 or 80 ◦ C above 15 percentage of 1-dodecanol (less pronounced swelling effect of individual globules and decreasing flow-through pores due to greater solvation by porogen). As in the case of synthesis performed at 60 ◦ C, for higher temperature values (70 and 80 ◦ C) an increase in permeability together with an increase in 1-dodecanol content (as a poor solvent) at

its lower concentrations is also observed, but this effect is shifted from %D = 15% (t = 60 ◦ C) to %D = 30% (for temperature of 70 ◦ C) and %D = 60% (for the temperature of 80 ◦ C). Further raising 1-dodecanol concentration contributed to a reduction of permeability which may be explained in the same way as in the case of polymerization performed at 60 ◦ C. Thus, the overlapping effect of temperature and porogen solvent composition indicates that there are no strict principles which enable the selection of proper polymerization conditions. The highest surface area was observed for the monolith synthesized at 80 ◦ C with 15% of 1-dodecanol in the porogen solvent (SBET = 39.6 m2 /g) but, in this case, the permeability was very low. From the practical point of view, it seems more favorable to perform the immobilization of the enzyme on the bed of higher permeability (lower surface area and back pressure) and to have the possibility of using a longer capillary. Therefore, the monolith synthesized at 80 ◦ C with 30% content of 1-dodecanol in the porogen was finally used as a support. The polymer obtained under the above mentioned conditions was characterized by lower surface area (5.5 m2 /g), but it showed high permeability (KF = 4.83 × 10−14 m2 ) and also a homogenous structure (there were no voids at the interface between the monolith and the capillary wall, (Fig. 4).

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Fig. 6. Comparison of mass spectra in the range of m/z covering the peptide chains with missed cleavage sites: (A) post-reaction mixture (classical digestion method, incubation time: 14 h) with marked signals of miscleaved polypeptides corresponding to sequences: 1: MDAKMYLGYEYVTAIR (oxided methionine), 2: WCAVSEHEATKCQSFR (carbamidomethylated cysteines) 3: LHDRNTYEKYLGEEYVK, (B) eluate from microreactor (F = 0.05 ␮l/min).

Fig. 4. SEM image of a monolithic capillary (i.d. 150 ␮m) used for microreactor preparation with the immobilized enzyme (1500×).

Fig. 5. Comparison of mass spectra: (A) solution of intact transferrin, (B) solution of transferrin passed through a microreactor (at the top−the spectra fragments at a higher m/z values; at the bottom, the spectra fragments in the range from 200 to 1600 Da).

3.2. Characterization of the prepared microreactor with regard to trypsin activity The obtained results indicated that the degree of BAEE hydrolysis decreased with the increasing flow rate as well as substrate concentration, which was not surprising (Table 1). The flow rate determined the residence time of the substrate molecules in the microreactor. In other words, the longer residence time the higher

the probability of contacting the substrate with the trypsin active site. On the other hand, when the concentration of BAEE solution is increased (at the constant flow rate), temporary saturation of the biocatalyst’s active sites caused the observed phenomenon. In the prepared microreactor, the 10 mM BAEE solution was completely hydrolysed at 0.75 ␮l/min, which suggests a higher system efficiency compared to the previously described ␮-IMER (less than 2 mM BAEE solution was converted into BA at the flow rate of 0.449 ␮l/min [16]). It has also been observed that the degree of BAEE hydrolysis increased with temperature and reached the highest value at 37 ◦ C (natural activity conditions for the enzymes). Higher temperature conditions were not tested as they could have an adverse effect on the longevity of the immobilized enzyme and might lead to loss of its enzymatic activity (due to protein denaturation). The situation has already been mentioned in literature [40]. The longevity of trypsin and durability of the enzymatic system are other important issues. In the course of all the chromatographic analyses, neither additional peaks on the chromatograms were recorded nor the baseline was affected by the elution of unexpected compounds from the column (which would suggest the detachment of trypsin and leaching it out from the support). Additionally, after 12 days of daily microreactor use, the hydrolysis of the 25.00 mM BAEE solution (carried out under the same conditions) presented almost unchanged performance (37.02 and 37.00%, respectively). It indicates the fact that, during this time period, trypsin retained its activity. However, it should be noted here that, after performing the experiments, the microreactor was flushed with a storage solution containing NaN3 (for 30 min, at the flow rate of 1 ␮l/min) at the end of the working day and stored at 4 ◦ C overnight. In order to verify the reproducibility of the elaborated method, another microreactor (a capillary of 8.0 cm in length, referred to as ␮-IMER 2) was prepared, and then, the 10.0 mM BAEE solution was passed through the ␮-IMER 2 at the flow rate of 1.0 ␮l/min, at room temperature. The obtained degree of hydrolysis was 93.35% (three measurements were performed, RSD = 0.30%). The initial length of the first microreactor (␮-IMER 1) was also 8.0 cm, but cutting its ends was required as they were crushed by the ferrules during equipment assembling. The hydrolysis degree obtained for 10.00 mM BAEE solution was 83.55% (Table 1, RSD = 3.40%, n = 3) while the microreactor length was 7.7 cm. The normalized results expressed by the hydrolysis degree per one centimeter of the capillary for ␮-IMER 1 and ␮-IMER 2 were 10.85 [%/cm] and 11.67

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Table 1 Comparison of peaks area of substrate before and after passing BAEE solution through a microreactor. ABAEE.st a

CBAEE.st a

ABAEE b

ABA b

50.346 257.189 626.729 1268.802 1544.716 1890.827

2 10 25 50 62.5 75

0 43.281 396.487 908.162 1238.398 1619.477

49.845 215.112 258.859 352.734 311.351 294.393

257.189 626.729 1268.802 1544.716 1890.827

10 25 50 62.5 75

0 316.036 807.998 1147.228 1433.531

626.729 1268.802 1544.716 1890.827

25 50 62.5 75

626.729 1268.802 1544.716 1890.827

25 50 62.5 75

b

CBAEE b

%H

F

49.845 258.393 655.346 1260.896 1549.749 1913.870

0 1.645 15.744 36.168 49.350 64.562

100 83.55 37.02 27.66 21.04 13.92

1

261.852 367.862 403.537 380.558 412.574

261.852 683.898 1211.534 1527.785 1846.105

0 12.533 32.170 45.711 57.139

100 49.87 35.66 26.86 23.81

0.75

181.037 642.786 1026.841 1366.499

441.240 466.771 511.594 434.605

622.277 1109.556 1538.436 1801.104

7.144 25.575 40.906 54.464

71.42 48.85 34.55 27.38

0.5

0 412.406 705.924 1160.679

602.545 765.076 763.678 597.271

602.545 1177.482 1469.601 1757.950

0 16.379 28.096 46.248

100 67.24 55.05 38.34

0.25

ABAEE.st − peak area of the substrate [mAU.s]; CBAEE.st − concentration of standard BAEE solution before passing through a ␮-IMER [mM]; ABAEE − peak area of the substrate [mAU.s]; ABA − peak area of the product [mAU · s]; A − total peak area [mAU · s]; CBAEE − concentration of BAEE in solution after passing the standard solution through a ␮-IMER [mM]; %H − degree of hydrolysis calculated according to Eq. (2); F − flow rate [␮l/min]. a The results obtained during preparation of calibration curve (R2 = 0.9998). b The results obtained after passing BAEE solution through a ␮-IMER at different flow rates.

Fig. 7. The mass spectra fragments of the eluate from the ␮-IMER containing signals derived from tryptic polypeptides.

[%/cm], respectively. It should be taken into account here that the both compared assays were performed at room temperature (without careful temperature control) which could contribute to the observed differences. However, the obtained values are similar which suggests good method reproducibility. 3.3. Transferrin digestion and MALDI-TOF analysis For the evaluation of a dual-enzyme ␮-IMER activity towards proteins resistant to tryptic proteolysis, the transferrin solution was passed through the ␮-IMER 2 (L = 8 cm) using a syringe pump. It is well known that trypsin cleaves the polypeptide chain at the carboxyl side of lysine (K) and arginine (R) but not before proline (P). When compared with trypsin, chymotrypsin is considerably less specific, and it hydrolyzes peptide bonds mainly on the C-terminal side of the aromatic amino acids (that is phenylalanine, tyrosine, and tryptophan) as well as leucine and methionine (at a slower rate) [28]. In view of the fact that intact transferrin was not detected on the MS spectrum of the eluate, it seems that the protein was completely hydrolysed in the microreactor. Instead, a lot of other signals at

low m/z values (below 1600) were recorded. It indicates that the protein was degraded to a large number of low molecular weight polypeptide chains (Fig. 5). As it is commonly known, after tryptic proteolysis some sequences of amino acids occur in more than one of the peptide chains which still contain unhydrolyzed peptide bonds between arginine (R) or lysine (K) and subsequent amino acids (so-called missed cleavage sites). It is particularly disadvantageous in the case of a quantitative analysis based on a specific peptide fragment which should be generated during tryptic digestion. In comparison with the PMF spectrum of transferrin classically digested in a solution (with dissolved high-purity trypsin), no signals derived from the peptides with tryptic missed cleavage sites were recorded on the MS spectrum of the ␮-IMER eluate (see MDAKMYLGYEYVTAIR, WCAVSEHEATKCQSFR, LHDRNTYEKYLGEEYVK in Fig. 6). However, signals corresponding to some sequences present in the above peptides (LHDR (Fig. 8), VTAIR (Fig. 9)) and some other signals resulting from trypsin activity have also been noticed (examples are presented in Fig. 7). T1 signal can be attributed to the peptide chain without aromatic amino acids (DSSLCK) towards which chymotrypsin is most

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Fig. 8. Scheme of cleaving the polypeptide: FRDHMKSVIPSDGPSVACVK KLHDRNTYEKYLGEEYVK by both enzymes (T−trypsin, C−chymotrypsin) and signals of resulting fragments recorded on MS spectra of eluate.

active. It probably explains its preservation after the reaction. The T2 (APNHAVVTR) and T3 (SVIPSDGPSVACVK) signals occurrence represents the polypeptides without phenylalanine (F), tyrosine (Y), tryptophan (W), and also methionine (M) or leucine (L). According to Rivera-Burgos and Regnier [36], such digestion of transferrin using trypsin-immobilized reactor led to results different from those obtained by the classical method. Their research findings show that partially digested polypeptides are preserved more often in the case of digestion in the trypsin-immobilized reactor. Their results demonstrate that, in the case of ␮-IMER use, the T1sequence occurs in 4 polypeptides and it was not detected alone (different flow rates were used and it was a flow-independent phenomenon). It is worth noticing that the T3 sequence occurred individually and in several different polypeptides (due to the missed cleavage sites before and after the discussed fragment) and some of them also included the DHMK sequence. On the recorded MS spectrum of the eluate, both T1 and T3 sequences did not occur in other polypeptides within m/z range used. Instead of DHMK sequence, the signal derived from RDHMK peptide was recorded on the spectrum which was attributed to the chymotrypsin activity (peptide cleaving between F and R). It should be emphasized that FT-T signal corresponds to the LHDR sequence which is present in the polypeptide with missed cleavage sites recorded on the PMF spectrum (Fig. 8). On the other hand, on the mass spectrum obtained for the postreaction mixture (involving the classical method), the intensive signal corresponding to MYLGYEYVTAIR sequence (also included in a peptide with a missed cleavage site MDAKMYLGYEYVTAIR) was recorded (Fig. 9), but it did not occur on the mass spectrum of the ␮-IMER eluate. It was probably caused by the fact that this signal derived from the polypeptide containing three tyrosine residues, and it was also hydrolysed by chymotrypsin (to VTAIR polypeptide and other extremely short fragments (MY, LGY and EY)). In the case of MS spectrum of the eluate, the recorded signals result from chymotrypsin or trypsin activity and also a combined effect of both enzymes (Fig. 10). It proves that, during immobilization of trypsin, chymotrypsin was also bound on the support and it evidently takes an active part in the hydrolysis of the model protein resistant to the proteolysis. It is obvious that chymotrypsin disrupts the high specificity of trypsin but it also enables obtaining shorter fragments with greater efficiency and prevents from obtaining the missing cleavage sites. It is probably due to the fact that additional

Fig. 9. Comparison of mass spectra fragments (on the top: eluate from microreactor (F = 0.05 ␮l/min), at the bottom: post-reaction mixture (incubation time: 14 h)) with polypeptide of sequence MYLGYEYVTAIR which occurs only on the MS spectrum in the case of using high-purity trypsin (digestion in solution) but was not recorded on the MS spectrum of eluate from microreactor. On the right − fragment of MS spectrum with signal resulting from chymotrypsin activity (VTAIR) after hydrolysis MYLGYEYVTAIR to shorter polypeptides.

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Fig. 10. The fragment of transferrin sequence with marked cleavage sites (T−trypsin, C−chymotrypsin) and examples of signals corresponding to polypeptides resulting from trypsin and chymotrypsin activity (PMF−mass spectrum obtained after trypsin digestion in solution, ␮-IMER − MS spectrum of the eluate from the microreactor).

protein cleaving by chymotrypsin facilitates the access of trypsin to the places of its typical activity (R and K residues). Consequently, the trypsin- and chymotrypsin-based ␮-IMER application resulted in transferrin sequence coverage 41.8–56.4%. In comparison, the sequence coverage obtained for the classical protocol including in-solution digestion (using high purity trypsin) was only 17.8%. In other research, transferrin digestion was performed in 45% acetonitrile (using a microreactor with commercially available trypsin-beads) which resulted in 46% sequence coverage [35]. It indicates that a dual-enzymatic system involving inexpensive reagents may be an alternative method used for sufficient protein identification. 4. Conclusions The preparation of a highly efficient microreactor with a dualenzyme system was described. The prepared ␮-IMER showed high enzymatic activity towards both low and high molecular weight substrates which allowed carrying out the reaction in a short time (the time needed to collect the eluate was 14 times shorter than the incubation time required in the case of in-solution digestion) and at minimum cost, without a significant efficiency loss and no sample contamination risk. The high efficiency of BAEE hydrolysis results from trypsin activity while the significant complete degradation of transferrin is due to the combined effect of activity of the two enzymes immobilized in the capillary monolithic col-

umn. Hence, the prepared microreactor can be potentially applied for the removal of proteins from samples (when proteins are not target analytes) or fragmentation of target proteins (previously isolated from the mixture), including those which are proteolytically resistant (such as transferrin). Therefore, it may enable the investigation of post-translational modifications including glycosylation, phosphorylation, and the presence of lipid moieties of the known proteins by the MS analysis of the obtained fragments or use of two-dimensional CE. Additionally, the immobilization of trypsin and chymotrypsin in one microreactor would make it possible to investigate the effect of inhibitors on their individual activity under identical conditions (such as dual-enzymatic systems developed for adenosine deaminase and xanthine oxidase [43]) and to remodel the processes occurring in a human body. ␮-IMER can be used as a model system imitating the intestinal digestion of proteins or also for mimicking the processes occurring in the gastrointestinal tract (for example in connection with a pepsin-immobilized reactor simulating gastric processes). It may also be a highly efficient tool for studying of protein biotransformation and the fate of orally administered bioactive peptides, hormones, or other protein substances. The above described microreactor preparation procedure (elaborated using the low-cost reagent) may be easily repeated with trypsin of high purity grade which would lead to obtaining a highly efficient and specific enzymatic system.

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Acknowledgments This work was supported by Symfonia I grant No. 2013/08/W/N28/0070, Maestro-6, Preludium grants No.: 2013/11/N/ST4/01837 and 2013/11/N/ST4/01835 from the National Science Centre, Kraków, Poland.

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Please cite this article in press as: K. Meller, et al., Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.02.070