Microwave-Assisted Protein Digestion in a Plate Well for Facile

Sep 25, 2017 - EC/3D in the plate well has demonstrated its potential as a robust and facile tool for the development of an automated protein digestio...
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Microwave-Assisted Protein Digestion in a Plate Well for Facile Sampling and Rapid Digestion Hyeonil Kim,†,‡ Han Sol Kim,†,‡ Dabin Lee,§,‡ Dongwon Shin,§ Daeho Shin,∥ Jeongkwon Kim,*,§ and Jungbae Kim*,† †

Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea Department of Chemistry, Chungnam National University, Daejeon 34134, Republic of Korea ∥ Bio Medical Technologies Co., Ltd., Seoul 04598, Republic of Korea §

S Supporting Information *

ABSTRACT: Protein digestion is one of the most important processes in proteomic analysis. Here, we report microwaveassisted protein digestion in a plate well, which allows for facile sampling as well as rapid protein digestion based on the combination of highly stable enzyme immobilization and 3D printing technologies. Trypsin (TR) was immobilized on polystyrene-based nanofibers via an enzyme coating (EC) approach. The EC with stabilized TR activity was assembled with the 3D-printed structure in the plate well (EC/3D), which provides two separated compartments for the solution sampling and the TR-catalyzed protein digestion, respectively. EC/3D can effectively prevent the interference of sampling by accommodating EC in the separated compartment from the sampling hole in the middle. EC/3D in the plate well maintained its protein digestion performance under shaking over 160 days. Microwave irradiation enabled the digestion of bovine serum albumin within 10 min, generating the MALDI-TOF MS results of 75.0% sequence coverage and 61 identified peptides. EC/3D maintained its protein digestion performance under microwave irradiation after 30 times of recycled uses. EC/3D in the plate well has demonstrated its potential as a robust and facile tool for the development of an automated protein digestion platform. The combination of stable immobilized enzymes and 3D-printed structures can be potentially utilized not only for the protein digestion, but also for many other enzyme applications, including bioconversion and biosensors.

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for the protein digestion. Even though high enzyme stability and facile manipulation can be achieved by the combination of immobilized TR and various platforms, the automated protein digestion still requires tedious handling, particularly during the sampling step. As an example, we previously reported nanobiocatalytic enzyme stabilization, where TR was immobilized on polystyrene-poly(styrene-co-maleic anhydride) (PSPSMA) nanofibers (NFs) via an enzyme coating approach (EC). EC was prepared by covalent attachment (CA) of enzymes on the surface of PS-PSMA NFs and then forming thick coatings of enzymes via subsequent cross-linking (Figure S-1).13 EC successfully stabilized the activity of TR compared to its control of CA enzymes on PS-PSMA NFs with no crosslinking, and the long-term protein digestion using EC was successfully demonstrated.23−25 However, due to their amorphous morphology and suspended form in an aqueous solution, EC interferes with the injection and removal of sample solution by jamming with the pipet tips.

roteins play a key role in all the biological systems, and the elucidation of protein functions via proteomics is of great importance for a better understanding of biological systems.1−3 Proteomics has expanded its applicability to many different scientific fields such as molecular biology,4 oncology,5 and neurology,6 and its target of interests is being shifted from the biomarker discovery to many exciting fields, including diagnostics.7,8 In a typical bottom-up approach of proteomic analysis, proteins are digested into peptides by a protease such as trypsin (TR) prior to the MS analysis of peptides. Even though the protein digestion is critical for successful protein identification, the current practice of in-solution digestion is long, tedious, and difficult to automate.9 A variety of efforts have been dedicated to the development of a rapid,10 recyclable,11 and automated digestion system.12 During the last one decade, we have witnessed unprecedented successes of enzyme immobilization approaches in stabilizing the enzyme activities. Various solid carriers, including nanofibers,13 nanoparticles,14 porous silica,15 sol− gel silica,16 and membrane,17 have been used for the immobilization of TR.18 At the same time, various platforms, such as microfluidics,19 microchips,20,21 and well plates,22 have been introduced for the facile manipulation of immobilized TR © XXXX American Chemical Society

Received: June 7, 2017 Accepted: September 13, 2017

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DOI: 10.1021/acs.analchem.7b02169 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry

PSMA NFs were detached from collector and stored at room temperature. Trypsin Immobilization on Electrospun Nanofibers. The 2 mg aliquots of PS-PSMA NFs were treated by 50% (v/v) ethanol solution for their dispersion.23,29,30 Ethanol-dispersed PS-PSMA NFs were washed with 10 mM sodium phosphate buffer (pH 7.9) and incubated in 2 mL of 10 mg/mL TR solution under shaking (200 rpm) at room temperature for 30 min and on a rocker (50 rpm) at 4 °C for 2 h. TR molecules were covalently attached on PS-PSMA NFs via the reaction between the maleic anhydride groups of NFs and amino groups of TR (CA-TR/NFs, CA). The GA solution was added to the sample at the final concentration of 0.5% (w/v) GA for the preparation of enzyme coatings of trypsin on the PS-PSMA NFs (EC-TR/NFs, EC). For the case of CA sample, 10 mM sodium phosphate buffer (pH 7.9) was added instead of GA solution. Samples were incubated overnight on the rocker under 50 rpm at 4 °C and washed with 100 mM sodium phosphate buffer (pH 7.9). The samples were further treated with 100 mM Tris-HCl buffer (pH 7.9) for 30 min to cap the unreacted aldehyde groups. The samples were washed with 10 mM sodium phosphate buffer (pH 7.9) excessively, and stored at 4 °C until use. Preparation of 3D Structures with 3D Printer. 3D structures were designed by using a computer-aided design (CAD) software, Solidworks 2014 (Dassault Systèmes SolidWorks Corp., Vélizy-Villacoublay, France), and printed by using a 3D printer (Rokit Universe, Seoul, Korea). Acrylonitrile butadiene styrene (ABS) filament was used as a raw material. The temperature of nozzle and heating bed for 3D printing was set to be 230 and 130 °C, respectively. After the printing, 3D structures were cooled at room temperature. Activity Measurement of Immobilized TR with 3D Structures. Immobilized TR samples (CA and EC) were assembled with 3D structures and loaded in the wells of 24 well plate (CA/3D and EC/3D). The activities of CA/3D and EC/ 3D were measured by the hydrolysis of 0.6 mM L-BAPNA in 2 mL of 10 mM sodium phosphate buffer (pH 7.9) under shaking (200 rpm) at room temperature. The generation of product, pnitroaniline, was monitored by measuring the absorbance increase at 410 nm (A410) with a UV−vis spectrophotometer (Shimadzu, Kyoto, Japan). Enzyme activity was calculated from the time-dependent increase of A410. One unit of TR activity is defined by the amount of enzyme that hydrolyzes 1 μmol of LBAPNA per 1 min in 10 mM sodium phosphate buffer (pH 7.9). Protein Digestion in 3D Structure-Based TR Platform. A total of 2 mL of 10 mg/mL reduced and alkylated BSA in 50 mM ammonium bicarbonate buffer (pH 7.9) was added to a plate well with CA/3D or EC/3D, and the well plate was incubated at 37 °C under shaking (200 rpm) for 12 h. After the digestion, 100 μL of digested solution was taken from the well, and 1 μL of formic acid was added to stop the protein digestion. The solution of digested protein was purified with C18 spin column and analyzed by using a MALDI-TOF mass spectrometer (Voyager DE-STR; Applied Biosystems, Foster City, CA, U.S.A.). Sequence coverage and number of identified peptides were calculated from the MALDI-TOF mass spectrometer peak and mass results. The performance stabilities of CA/3D and EC/3D were checked by repeating the BSA digestion over time. After protein digestion, CA/3D and EC/ 3D were washed with 10 mM sodium phosphate buffer (pH

In this paper, we report the combinational approach of highly stable enzyme immobilization and 3D printing to develop a well plate-based platform for protein digestion. 3D structures were fabricated by using a 3D printer, which can be assembled with EC (EC/3D) in a plate well. 3D structures were designed to provide a sampling hole that is surrounded by the compartment with the EC sample. By that way, the facile sampling after protein digestion is doable by not being interfered with the EC sample at all (Figure 1). To expedite

Figure 1. (A) Schematic illustration for the combination of stabilized trypsin coatings on nanofibers and 3D-printed structure in a plate well. (B) Comparison of the sampling without and with 3D structure in the presence of EC in a plate well.

the protein digestion, microwave was irradiated as an external energy to EC/3D.26,27 Activity difference of EC/3D with different configurations of 3D-printed structures was studied, and facile sampling in the EC/3D assembly was also demonstrated. Protein digestion performance and stability were also evaluated by using bovine serum albumin (BSA) as a model protein. The effect of microwave irradiation on the protein digestion and the stability of EC/3D were both evaluated under the repeated BSA digestions.

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EXPERIMENTAL SECTION Chemicals and Materials. TR, BSA, glutaraldehyde (GA, 25%), N-benzoyl-L-arginine 4-nitroanilide hydrochloride (LBAPNA), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), acetone, poly(styrene-co-maleic anhydride) (PSMA, Mw = 224400; maleic anhydride content = 7 wt %), ethanol, guanidine hydrochloride (Guanidine-HCl), DL-dithiothreitol (DTT), iodoacetamide (IAA), formic acid, sodium phosphate monobasic, sodium phosphate dibasic, Tris-HCl, ammonium bicarbonate, and Amicon Ultra-4 centrifugal filter unit with PLGC Ultracel-PL membrane (10 kDa) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). A 24-well cell culture plate was purchased from SPL Life Sciences Co., Ltd. (Pocheon, Korea). Polystyrene (PS, Mw = 900000) was purchased from Pressure Chemical (Pittsburgh, PA, U.S.A.). Pierce C18 spin column was purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Synthesis of Electrospun Nanofibers. PS-PSMA NFs were synthesized via electrospinning method by following the protocol of Kim et al.28 Briefly, PS and PSMA were dissolved in THF solution at the mixing ratio of 2:1 (w/w). The PS-PSMA mixture solution was loaded into a plastic syringe. Electrospinning was operated by feeding the mixture solution at 0.5 mL/h under the voltage of 7 kV. Electrospun PS-PSMA NFs were collected on aluminum foil collector. The collected PSB

DOI: 10.1021/acs.analchem.7b02169 Anal. Chem. XXXX, XXX, XXX−XXX

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6.11 × 10−2 units, respectively (Figure 2B). When 3D structures were introduced, the TR activity was reduced regardless to the configuration of 3D structures, compared to that of without 3D structure. It can be explained by the mass transfer limitation, which was inevitable in the presence of 3D structure in a plate well. However, facile sampling is allowed by the sampling hole of 3D structures. The activity of structure 2 was 1.7× higher than that of structure 1. The lower activity of structure 1 can be explained by the reduced volume of internal space due to additional bottom and side walls. By not having these bottom and side walls, structure 2 can provide more space for effective shaking of EC, which can increase the frequency of collision between the L-BAPNA substrates and immobilized trypsin molecules, leading to the improved activity. Interestingly, the activity of structure 3 was 3.1× higher than that of structure 2. The only difference between structure 2 and structure 3 was a meshed top, while structure 3 had a blocked top with no mesh. The higher activity of structure 3 than structure 2 can potentially originate from the modified hydrodynamic behavior within the plate well. Structure 2 with the meshed top cover would release the upward fluid momentum through meshes. On the other hand, structure 3 with the top cover with no mesh would redirect the upward fluid momentum to the downward one, which would allow for more rigorous shaking in the fluid under the top cover and increase the chances for the L-BAPNA substrates to be contacted with EC in the plate well. To check this hypothesis, we designed and performed an experiment by reducing the volume of fluid in the plate well. When 2 mL of L-BAPNA solution was added to the EC/3D, the meniscus of the solution was located above the 3D structure whose height was 11 mm. The volume of solution above 3D structure corresponded to 0.6 mL. The EC activities of structures 2 and 3 were measured after adding 1.4 mL of LBAPNA solution, which allows for the meniscus of fluid to touch the top cover of the 3D structures. The measured EC activities with structures 2 and 3 were 3.80 × 10−2 and 7.59 × 10−2 units, respectively (Figure S-2), representing 1.9× and 1.2× higher activities than those with 2 mL of L-BAPNA solution, respectively. The EC activities with structure 3 were less affected by the solution volume than those with structure 2. In other words, more L-BAPNA solution below the top cover of structure 3 participated in the EC-catalyzed hydrolysis of LBAPNA. This result suggests that structure 3 improves the fluidic momentum due to the counteract of the top cover with no mesh against the upward fluid momentum. Protein Digestion in a Plate Well with 3D Structure. BSA was used as a model protein to demonstrate the protein digestion in a plate well with EC/3D. Among three different structures to be tested, structure 3, allowing for the highest EC activity, was selected for the further experiment of protein digestions. EC was assembled with the structure 3 (EC/3D) in a plate well. Reduced and alkylated BSA was added to the plate well with EC/3D, and digested under shaking (200 rpm) at 37 °C for 12 h. After the protein digestion, an aliquot of digested solution was analyzed via MALDI-TOF MS. The stabilities of CA/3D and EC/3D were investigated by checking their protein digestion performance over time (Figure 3). After each digestion, CA/3D and EC/3D were washed five times with fresh buffer and incubated under continuous shaking (200 rpm) at room temperature. It was confirmed that no residual peptides were observed in the plate well after four washings (Figure S3).

7.9) and stored at room temperature under shaking (200 rpm) until the next use. Protein Digestion under Microwave Irradiation. Microwave was irradiated at 400 W by using the microwave irradiation system (ASTA Inc., Suwon, Korea). A total of 2 mg of 10 mg/mL reduced and alkylated BSA was added to the EC/ 3D and microwave-assisted protein digestion was carried out under the controlled temperatures of 25, 37, and 55 °C. After microwave-assisted protein digestion, 100 μL of solution was taken from the well through the sample hole of 3D structures, and treated with 1 μL formic acid to stop the protein digestion. The solution was analyzed by MALDI-TOF MS as described in the previous section.

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RESULTS AND DISCUSSION 3D Structure in a Plate Well. 3D structures with three different configurations (structures 1, 2, and 3) were prepared by using a 3D printer. Each structure was assembled with EC and loaded in a plate well (Figure 2A). The height and diameter

Figure 2. (A) Pictures of EC without 3D structure and with structures 1, 2, and 3 in a plate well (from left to right). (B) Activities of EC without 3D structure and with structures 1, 2, and 3. Inset images represent the 3D structure in each plate well.

of 3D structures were 11 mm and 16 mm, respectively. All the 3D structures were basically designed to have a hollowed cylindrical hole (4 mm diameter) in the middle, to allow for easy insertion and removal of a pipet tip for the facile addition of protein solution and sampling of digested peptide solution together with many follow-up washings for the next step digestion. This sampling hole was surrounded by the compartment that accommodates the immobilized TR on PSPSMA NFs (CA and EC). For the case of structures 1 and 2, both sampling hole and compartment were composed of mesh structure. The intermesh distance was 0.8 mm, which provides enough space for the easy fluid passage. The top cover of structure 3 did not have mesh, while the sampling hole was fabricated in a meshed structure. EC activity in a plate well was measured by the hydrolysis of L-BAPNA in the 2 mL of 10 mM sodium phosphate buffer (pH 7.9) under shaking (200 rpm). The activity of EC without 3D structure was 12.5 × 10−2 units, which is about 13.2% of free TR activity. The reduced TR activity after the immobilization could be due to the loss of unbound TR during the washing step in the EC preparation. The activities of ECs assembled with structures 1, 2, and 3 were 1.12 × 10−2, 1.98 × 10−2, and C

DOI: 10.1021/acs.analchem.7b02169 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 3. Sequence coverage (A) and number of identified peptides (B) from the iterative BSA digestions in the plate well with CA/3D and EC/3D for 164 days.

CA/3D showed a very rapid drop in its protein digestion performance within 2 days. For example, the sequence coverage and identified peptide number decreased to 2.7% and 3, respectively, within 2 days, both of which are too low for the identification of BSA. On the other hand, EC/3D maintained high level of protein digestion performance after 164 days of repeated uses at 37 °C and storage at room temperature under continuous shaking (200 rpm). This high stability of EC/3D can be explained by the cross-linked TR molecules, which effectively prevent the denaturation of TR molecules. In addition, the coating of cross-linked TR molecules on the surface of PS-PSMA NFs is also resistant to the autolysis of TR.13 Microwave-Assisted Protein Digestion for Rapid Digestion. To expedite the protein digestion, microwave was applied as an external energy to the EC/3D in the plate well at three different temperatures of 25 °C, 37 and 55 °C (Figure 4). Microwave-assisted protein digestion at 37 °C for 10 min resulted in 75.0% and 61 of sequence coverage and number of identified peptides, respectively (Figure 5). These results show that protein digestion was effecitvely expedited under micro-

Figure 5. Sequence coverage (A) and number of identified peptides (B) from the BSA digestion in the plate well with EC/3D under microwave irradiation at three different temperatures (25, 37, and 55 °C).

wave irradiation, which are comparable with those obtained by 12 h digestion without microwave irradiation. At 25 °C, on the other hand, the sequence coverage and number of identified peptides were 13.5% and 11 after 10 min digestion, respectively. Improved performance of protein digestion at higher temperature of 37 °C rather than 25 °C can be explained by the Arrhenius equation where the rate of chemical reaction exponentially proportional to the temperature because of the increased kinetic energy.31 However, microwave-assisted protein digestion at 55 °C showed decreased performance compared to 37 °C. It can be elucidated by the counterbalance of heat-induced kinetic activation with the denaturation of enzyme structure. It would be worth to note that microwaveassisted protein digestion showed increased performance compared to the protein digestion without microwave irradiation (Figure S-4). It implies that microwave irradiation achieved effective and rapid heating of local system by directly applying its electromagnetic energy to the reaction solution.32 To investigate the stability of EC/3D under recycled uses, the BSA digestion was performed under microwave irradiation at 37 °C over time. After each digestion, EC/3D was washed and reused for the next BSA digestion in the same condition. The BSA digestion performance of EC/3D under microwave irradiation was fluctuating very rigorously, but maintained the sequence coverage more than 30% and identified more than 20 peptides even under the recycled uses for 30 times (Figure 6), which are generally regarded as good performance of protein digestion. The data fluctuation can be explained by the nonshaking condition of microwave irradiation system, which would create the heterogeneity of fluidics leading to varied performance at each BSA digestion. Interestingly, however, the performance of protein digestion, represented both by sequence coverage and number of identified peptides,

Figure 4. Schematic illustration for the protein digestion under microwave irradiation. D

DOI: 10.1021/acs.analchem.7b02169 Anal. Chem. XXXX, XXX, XXX−XXX

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Technical Note

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b02169. Supporting figures for scheme, activity study, washing effect, and protein digestion without microwave (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jungbae Kim: 0000-0001-8280-7008 Author Contributions ‡

These authors contributed equally to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Global Research Laboratory Program (2014K1A1A2043032) and Nano·Material Technology Development Program (2014M3A7B4052193) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT. This work was also supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded by Korea Small and Medium Business Administration in 2015 (C0298433). This research was also supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (No. 20142020200980). The authors would like to thank Eunjung Son for her help in the revision process.

Figure 6. Sequence coverage (A) and number of identified peptides (B) from the iterative BSA digestions under microwave irradiation at 37 °C for 10 min.

maintained the reasonably good digestion results even though both of them fluctuated. This result reveals that the protein digestion under microwave irradiation is good enough to maintain the decent performance by inducing the accelerated motion of water molecules under the microwave irradiation, which potentially contribute to the local mixing.32

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CONCLUSIONS By the combination of highly stable enzyme immobilization in the form of enzyme coatings (EC) and 3D printing technologies (EC/3D), we developed an efficient platform of protein digestion in a plate well under microwave irradiation, which potentially enables automated, fast and stable system of protein digestion with an option of facile sampling. In EC/3D, covalent attachment of trypsins on nanofilbers and cross-linking of additional trypsins were utilized to prepare thick trypsin coated nanofibers, which were then assembled with a 3D structure in a plate well. EC/3D in plate well not only allowed the facile sampling but also showed long-term stability by maintaining most of its initial protein digestion performance under shaking for 164 days. Microwave irradiation expedited and improved the protein digestion by generating the results of 75.0% sequence coverage and 61 identified peptides within 10 min of BSA digestion. EC/3D in the plate well has demonstrated its potential as a robust and facile tool for the development of an automated protein digestion platform. The combination of stable nanobiocatalysts and 3D-printed structures can be potentially utilized not only for the protein digestion, but also for many other enzyme applications including bioconversion and biosensors. E

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DOI: 10.1021/acs.analchem.7b02169 Anal. Chem. XXXX, XXX, XXX−XXX