In Situ Caging of Biomolecules in Graphene Hybrids for Light

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In Situ Caging of Biomolecules in Graphene Hybrids for Light Modulated Bioactivity Gong Cheng, Xiaohui Han, Si-Jie Hao, Merisa Nisic, and Si-Yang Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17544 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Applied Materials & Interfaces

In Situ Caging of Biomolecules in Graphene Hybrids for Light Modulated Bioactivity Gong Cheng, †,‡ Xiao-Hui Han, † Si-Jie Hao, † Merisa Nisic, † and Si-Yang Zheng*,†,‡ †

Department of Biomedical Engineering and

‡

Material Research Institute, The Pennsylvania State

University, University Park, Pennsylvania 16802, United States KEYWORDS: graphene, near-infrared light, compartment, nanoreactor, biomolecules ABSTRACT: Remote and noninvasive modulation of protein activity is essential for applications in biotechnology and medicine. Optical control has emerged as the most attractive approach owing to its high spatial and temporal resolutions; however, it is challenging to engineer light responsive proteins. In this work, a near-infrared (NIR) light-responsive graphene-silica-trypsin (GST) nanoreactor is developed for modulating the bioactivity of trypsin molecules. Biomolecules are spatially confined and protected in the rationally designed compartment architecture, which not only reduces the possible interference but also boosts the bioreaction efficiency. Upon NIR irradiation, the photothermal effect of GST nanoreactor enables the ultrafast in situ heating for remote activation and tuning of the bioactivity. We apply the GST nanoreactor for remote and ultrafast proteolysis of proteins, which remarkably enhances the proteolysis efficiency and reduces the bioreaction time from the overnight of using free trypsin to seconds. We envision that this work not only provides a promising tool of ultrafast and remotely controllable proteolysis for in vivo proteomics in study of tissue microenvironment and other biomedical applications but also paves the way for exploring smart artificial nanoreactors in biomolecular modulation to gain insight in dynamic biological transformation.

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1. INTRODUCTION Proteins play indispensable roles in living organisms by regulating most chemical reactions involved in numerous biological processes.1, 2 Tremendous efforts have been made to decode their structures and understand their functions.3,

4

However, understanding the temporal and

spatial activity of individual proteins in the biological system and their dynamic functions in various biological processes is still a great challenge, due to the absence of effective strategies to accurately and dynamically control bioactivity of proteins.5, 6 Mimicking the complicated natural system of cell, the design and construction of the smart artificial nanosystem that can protect the proteins and tune their bioactivity have drawn much attention.7-10 For instance, pH-responsive and temperature-sensitive compartments were explored to control the bioactivity of enzymes for on-demand biocatalysis and gene-directed protein synthesis.11,

12

However, alteration of the

systemic and surrounding pH or temperature would limit the extensive application of proteins in various life science and biotechnology. Thus, it is critical to control the activity of biofunctional proteins remotely and spatiotemporally by using an external stimulus.13-15 Optical control of bioactivity of proteins has sparked tremendous interest as the most attractive strategy for the stimulus-responsive system due to its high spatial and temporal resolutions.16 To enable the light responsiveness, proteins of interest are engineered by either genetical fusing with photosensitive protein domains or chemical modification of photocleavable/photoswitchable moieties.17, 18 However, most of these tailored proteins are responsive to ultraviolet or visible light which has limited tissue penetration but considerable phototoxicity. NIR light is an ideal light source for deep tissue penetration and in vivo application.19,

20

Recently, artificial

nanosystem assisted optical activation of transforming growth factor β signaling demonstrated that the photothermal effect of carbon nanotube included in the nanosystem could disrupt the


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association of the molecular interaction, resulting in the release of the transforming growth factor β from the artificial nanosystem to activate the downstream signal transduction in live cells and modulate cellular function.21 Although promising, modification of proteins to adapt the conjunction on carbon nanotube is indispensable, which may sacrifice the protein bioactivity and result in unpredicted issues in practical applications.22,

23

Furthermore, the requirement for

engineering additional NIR light-responsive association proteins compromises the extension of this strategy in biotechnology. Tissue microenvironment is critical in various aspects of in vivo biological processes, and the use of proteomics to study tumor microenvironment not only elucidates the underlying mechanisms,24,

25

but also leads to biomarker discovery from blood.26,

27

However, tissue

sampling is invasive and can only capture a snapshot of the proteome. Degradation and cleavage proteins in tissue can generate small fragments to enter the blood passively, producing informative traces of tissue microenvironment.28 Proteolytic enzyme (e.g. trypsin) can interact with other proteins and cleavage proteins to peptides for proteomics. However, modulation of the bimolecular activity of trypsin is a great challenge. Therefore, a remotely controllable nanosystem that can realize in situ proteolysis in deep tissue is highly desirable. Herein, we report the design and demonstration of a bio-mimic nanoreactor that is capable to perform proteolysis remotely controlled by NIR irradiation. We constructed hierarchical graphene-silica nanostructures with compartment architecture, and bioactive trypsin was precisely assembled in separate domains to form graphene-silica-trypsin nanohybrids (GST) as the NIR light-responsive artificial nanoreactor (Scheme 1). Remote activation of the artificial nanoreactor under NIR light radiation can lead to rapid in situ heating and consequent modulating the protein activity. We



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further explored the utility and potential of NIR remotely controllable GST nanoreactor for practical application in high throughput proteomics and in vivo proteomics.

Scheme 1. Schematic illustration of (A) the construction of the GTS nanoreactor and (B) ondemand modulation of trypsin activity by NIR radiation for ultrafast proteolysis. 2. EXPERIMENTAL SECTION Reagents

and

Materials.

cetyltrimethylammonium

Graphite

powder,

bromide (CTAB),

ammonium

ammonium

bicarbonate

(NH4HCO3),

nitrate (NH4NO3), fluorescein

isothiocyanate (FITC), 1,3,5-trimethylbenzene (TMB), trifluoroacetic acid (TFA), acetonitrile (ACN), phosphoric acid (H3PO4), hydrogen peroxide (H2O2), sulfuric acid (H2SO4), hydrochloric acid (HCl) and ethanol (EtOH) were purchased from Alfa Aesar. Potassium permanganate (KMnO4), Tetraethylorthosilicate (TEOS), 2,5-Dihydroxybenzoicacid (2,5-DHB), dopamine (DA), trypsin (from bovine pancreas, TPCK treated), bovine serum albumin (BSA), betalactoglobulin, myoglobin and cytochrome c (Cyt c) were purchased from Sigma-Aldrich (St.


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Louis, MO, USA). The pork was purchased from a local grocery store. All the chemical agents were used without further purification. Preparation of graphene-silica nanostructure. Graphene oxide (GO) was synthesized from natural graphite powder by a modified Hummers’ method.29-31 Assembly of porous silica on graphene oxide was realized using an advanced Stöber method.32, 33 Briefly, prepared GO (40 mg) was fully dispersed in 500 mL of deionized water with 1.12 g of CTAB. Then, 6.0 mL NaOH solution (0.1 M) and 94.0 mL deionized water were added to the solution, followed by stirring for 30 minutes at 60 °C. Next, 5.6 mL of a TEOS/ethanol solution (v/v: 1/4) were injected into the homogeneous solution, and intense agitation was applied for 2 minutes. The mixture obtained was heated to 60 °C for 12 hours. The resulting product, graphene oxide silica (GOS), was separated by centrifugation and then washed with deionized water and ethanol. The graphene-silica nanostructure with large nanopores was prepared according to a swelling agent incorporation method with a slight modification.34 Briefly, the as-synthesized GOS (0.5 g) was well dispersed in ethanol (15.0 mL) by sonication, followed by the addition of 30.0 mL of the mixture (v/v: 1/1) of deionized water and TMB. The mixture was transferred to the autoclave and kept still for 24 hours at 140 °C. The resulting black powder was washed with ethanol and deionized water for several times. To remove the pore-generating template, the black powder was transferred to an ethanol solution (120.0 mL) containing NH4NO3 (0.5 g) and stirred at 60 °C for 3 hours. This step was repeated twice. After further washing with deionized water and ethanol, the obtained products (GS) were dried under vacuum. Construction of GST nanoreactor. GS nanostructures were first activated via a bio-mimic strategy. GS nanostructures (100.0 mg) were fully dispersed in 50.0 mL of 20.0 mM Tris-HCl buffer (pH=8.0) by sonication for 30 minutes. Then, 50.0 mL of dopamine solution (1.0 mg mL-



1

) was slowly injected into the GS suspension under continuous sonication for half an hour. The

products were collected and washed several times with deionized water and redispersed into deionized water. FITC labeled trypsin (trypsin-FITC) was prepared according to a reported method with slight modification.35 Trypsin solution (1.0 mL, 2.0 mg mL-1 in 0.1 M sodium carbonate buffer, pH=9.0) and 50.0 µL of 1.0 mg mL-1 FITC solution (in anhydrous DMSO) were gently mixed and incubated at 4 °C overnight. After that, the product was spin-dialyzed through 3 kDa cut-off filter (Millipore), and washed three times with 25.0 mM sodium carbonate and twice with NH4HCO3 buffer. The construction of GST was easily conducted at room temperature. Typically, 1.0 mL of activated GS (1.0 mg mL-1) and 1.0 mL of trypsin or trypsinFITC (dissolved in 50.0 mM NH4HCO3 with designed concentrations) were mixed with a vortexer for 2 hours. Subsequently, the GS nanostructure loaded with trypsin molecules was collected and washed three times with the NH4HCO3 buffer to remove any weakly bound trypsin. The amount of trypsin immobilized was measured using BCA Protein Assay Kit. The obtained products were further mixed with 1.0 mL of arginine (0.5 mg mL-1) solution followed by purification with the NH4HCO3 buffer. The obtained GST was dispersed into the NH4HCO3 buffer (10.0 mg mL-1) via vortexing to form a homogeneous suspension. The photothermal response of the GST nanoreactor. Tests of the photothermal response of the GST were conducted at room temperature. The laser spot size was adjusted and kept at 0.5 cm in diameter using a lens. 50.0 µL of the NH4HCO3 buffer with certain amounts (0, 5, 10, 25, 50, 100 µg) of the GST was irradiated by NIR laser with designated power. The temperature of the solution was recorded every 10 seconds by an infrared thermometer. Cell culture and protein extraction. Human breast cancer cell line MDA-MB-231 was cultured in high glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), 100


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units per mL penicillin and 100.0 mg mL-1 streptomycin. Cell culture was maintained in humidified incubators at 5% CO2 and 37 °C. At 70-80% confluence, cells were harvested from culture flasks by incubation with 0.5% Trypsin-EDTA, washed with Dulbecco’s phosphate buffered saline and used in subsequent experiments described below. To extract the protein, cultured cells were first washed with 5.0 mL of DPBS for twice, and then 1.5 mL ice-cold RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail was added and followed by incubation in a 4 °C refrigerator for 5 minutes. After that, the treated cells were collected using a cell scraper and transferred into a tube on ice for 5 minutes incubation. The obtained cell lysate was centrifuged at 8000 g under 4 °C for 10 minutes. The supernatant was collected, and the final protein concentration was determined. Proteolytic catalysis of proteins using the GST nanoreactor. The proteins (bovine serum albumin, myoglobin, beta-lactoglobulin or cytochrome c) were dissolved into 50.0 mM of NH4HCO3 buffer to form protein solutions (0.1 mg mL-1). NIR light-induced proteolytic catalysis was conducted at room temperature. Briefly, the above protein solution (50.0 µL) was well mixed with a certain amount of GST nanoreactor, and the mixture was irradiated by a NIR laser with adjustable power for several seconds. Subsequently, 1.0 µL acetic acid was added to the mixture, followed by centrifugation, and the supernatant was collected for proteomic detection. For comparison, proteolytic catalysis of proteins using the conventional strategy of enzymes in solution was also conducted. Typically, trypsin was then added to the protein solution with an optimized enzyme/substrate ratio, and the solution was incubated at 37 °C for 1 minute or overnight. The subsequent procedure is similar to the previous description. For regeneration, the collected GST composites were washed with the NH4HCO3 buffer three times. To test the stability, GST and free enzyme were kept at room temperature for a month, and samples were



taken out at different intervals for proteolytic catalysis as described above. For whole cell proteolysis, the extracted proteins from cell line MDA-MB-231 were purified, denatured, and reduced via a universal sample preparation method.36 The digestion process was similar to that of standard proteins described earlier, and the NIR laser radiation was repeated for three times. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) analysis. 1.0 µL of proteolytic digest was mixed with 1.0 µL of matrix solution containing 20.0 mg mL-1 DHB (in 50% acetonitrile aqueous solution, v/v) and 1% (v/v) H3PO4 aqueous solution by pipetting. 0.5 µL of the mixture was deposited onto the MALDI target. MALDI-TOF mass spectrometry analysis was performed on a MALDI-TOF/TOF mass spectrometer (AB SCIEX 5800, Foster City, CA, USA) in positive ion mode with a 355 nm Nd:YAG laser, 200 Hz repetition rate, and 20 kV acceleration voltage. Search parameters of fragment ion spectra were submitted to MASCOT (http://www.matrixscience.com/) for a database search and identification of corresponding peptides. Liquid chromatography-mass spectrometry (LC-MS) analysis. An AB SCIEX TripleTOF 5600 System (Foster City, CA, USA) equipped with an Eksigent nanoLC Ultra and ChiPLCnanoflex (Eksigent, Dublin, CA) in Trap Elute configuration was employed for LC-MS measurement. The acquired mass spectrometric raw data was processed using ProteinPilot 5.0 software (AB SCIEX) with the Paragon search mode. The ProteinPilot Descriptive Statistics Template (PDST, AB SCIEX) was used for alignment of multiple results and evaluation of false discovery rate (FDR). Characterization. Scanning electron microscopy (SEM) images were obtained on a field emission scanning electron microscope (FESEM; NanoSEM 630, NOVA). Transmission electron


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microscopy (TEM) images were taken with a JEOL-2010 microscope at the accelerating voltage of 200 kV. Small-angle X-ray scattering (SAXS) patterns were collected on a PANalytical Empyrean X-ray powder diffractometer with a Scatter-X attachment, and the detective range was from 0.5 to 5 degrees. UV-Vis spectra were recorded on a Perkin-Elmer Lambda 950 UV-VisNIR Spectrophotometer. Fluorescence intensity was detected using a SpectraMax® M5 Microplate Reader from Molecular Devices LLC. Fourier transform infrared spectra were determined on a Bruker Vertex V70 FTIR spectrometer and scanned from 400 to 4000 cm-1 at a resolution of 6 cm-1. Raman spectra were recorded on a WITec Confocal Raman instrument with a 480 nm laser wavelength. Nitrogen adsorption isotherms were measured at a liquid nitrogen temperature (77 K) with a Micromeritcs ASAP 2020 apparatus. The specific surface area was determined by the Brunauer-Emmett-Teller (BET) method. The total pore volume was evaluated by the t-plot method, and pore size distribution was analyzed with the supplied BJH software package from the adsorption branches of the isotherms. Microscopy images were obtained on a fluorescence microscopy (Olympus IX71). The temperature change was recorded by an infrared thermometer (Fluke, USA) at designated time intervals. A continuous wave diode laser (DS311322-110-K808F14CD, BWT Beijing Ltd., China) with a wavelength of 808 nm was used for the laser irradiation experiments. 3. RESULTS AND DISCUSSION Construction and Characterization of GST nanoreactor. Scheme 1 illustrates the construction of GST nanoreactor. Graphene oxide (GO; Figure S1) nanosheets were prepared as the backbone for assembly of hierarchical porous silica nanostructure to form a sandwich-like graphene-silica (GOS; Figure 1A-C) nanosheets. The transmission electron microscopy (TEM; Figure 1B) and N2 adsorption-desorption isotherm analysis (Figure 1G) indicates the presence of



many uniform nanopores in the nanosheets with pore size focus on ~3.1 nm (Figure 1G inset), which is too small to fill the trypsin molecules (size: ~3.5 nm).37 To reduce the graphene oxide core and to generate the 3D silica nanostructure with hierarchical porous structure, GOS nanosheets were treated using the swelling agents by a solvothermal approach to form the graphene-silica (GS) nanohybrids with reorganized porous structure. The black color of GS (Figure 1F) indicates the GO backbone was reduced during the solvothermal treatment, which is further supported by the redshift of the absorption peak in UV-Vis spectra (Figure 1H) and the increase of the intensity ratio of the D band and G band (ID/IG) in Raman spectra (Figure S2). Under the solvothermal conditions, the strong hydrophobic interaction between surfactants’ alkyl chain and TMB would lead to the incorporation of TMB molecules in the mesopores,38 while part of silica dissolved in the water/ethanol mixture and redeposited around the reorganized templates. Thus, different from the morphology of GOS nanosheets, GS nanosheets have the rough surface and unique hierarchical porous structure (Figure 1D, E). N2 adsorption-desorption isotherm analysis indicates that GS presents apparent hysteresis loops of type H2 (Figure 1G), revealing the mesoporous structure with 3D cage-like nanocompartments and interconnected pores.39, 40 The BET surface areas of GOS and GS are 578.8 and 303.6 m2 g-1, respectively. However, the pore volume of the GS nanosheets (~0.73 cm3 g-1) is slightly higher than that of GOS (~0.64 cm3 g-1), indicating the remained porous structure. Nanopores with various sizes were derived from the cavity and 3D network structure with two major pore sizes of ~3.5 and ~8.0 nm (Figure 1G inset). Notably, the water-solubility of the GS nanosheets is excellent (Figure 1F), due to the presence of hydrophilic functional groups on silica (Figure S3).


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Figure 1. Morphology and structure of graphene-silica nanostructures. (A, D) SEM, (B, E) TEM and (C, F) water dispersion images of GOS (A-C) and GS (D-F) nanosheets. (G) N2 adsorptiondesorption isotherm and pore size distribution (inset) of GOS and GS nanosheets. (H) UV-Vis spectra of GO, GOS and GS nanosheets. The next step is to cage trypsin into the hierarchical GS nanostructures at room temperature by a bio-adhesive self-assembly (Figure S4).41,

42

Time-dependent loading curve (Figure 2A) of

trypsin indicates that trypsin molecules can be trapped rapidly into the nanocompartments due to the unique hierarchical porous structure of GS. In addition, the density of trypsin molecules assembled in GS nanostructure can be controlled by tuning the initial concentration (Figure 2B). Successful construction of the graphene-silica-trypsin (GST) nanoreactor was confirmed using fluorophore FITC labeled trypsin (trypsin-FITC). As displayed in fluorescence microscopy images (Figure 2C), the GST nanoreactor fluoresced after the assembly of trypsin-FITC. As



further analyzed by Fourier transform infrared spectroscopy (FTIR), in comparison to those of the activated GS nanostructures and pure trypsin, several characteristic absorption peaks of trypsin could be observed in the range of 1000-1800 cm-1 (C=O stretching and N-H bending) and 2750-3000 cm-1 (N-H stretching)43, 44 in FTIR spectrum of the GST nanoreactor (Figure 2D), indicating the successful assembly of trypsin in the GS.

Figure 2. Construction of GST nanoreactor. (A) Adsorption and self-assembly of trypsin into the GS nanostructures as a function of time. (B) Control the density of assembled trypsin in GST by varying the initial concentration. (C) Fluorescence microscopy images of GST nanoreactor with assembled trypsin-FITC. (D) FTIR spectra of activated GS, Trypsin, and GST nanoreactor. GST nanoreactor is characterized by their NIR photothermal responsiveness, owing to the rational incorporation of graphene nanosheet as the core. As displayed in Figure 3A, the temperature of GST solution with GST nanoreactor under radiation of NIR laser can quickly


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ramp to 50 °C in 40 seconds; however, no apparent temperature increase was observed for the pure aqueous solution or silica solution under the same radiation. These results indicate that the graphene backbone is critical for rapid boosting the in situ temperature of GST nanoreactor. As the temperature is important for efficient proteolysis by trypsin, we then carefully investigated the photo-thermal effect of GST nanoreactor under NIR radiation with different GST amount, exposure time and laser power to optimize the GST activation (Figure 3B). Under NIR radiation, the temperature of GST solution ramps rapidly at the beginning while stabilizes with the increase of radiation time. Increasing the amount of the GST or the laser power would also promote the temperature ramping speed. Importantly, the GST nanoreactor has a wide temperature window located in the optimal range for trypsin activity (35-50 °C).

Figure 3. NIR photothermal response of GST nanoreactor. (A) Thermal images and photos of GST suspension, silica suspension, and water after NIR radiation of 2 W for 40 seconds. (B) NIR photothermal response of GST nanoreactor at different doses, NIR radiation time and NIR laser power.



GST Nanoreactor for Rapid and Remote Modulation of Trypsin Biomolecules. The ability to reliably identify and accurately quantify any protein targets in the proteome is an essential task in life sciences research and a fundamental approach in precise clinical diagnosis.45 Mass spectrometry (MS)-based proteomics (digestion of target proteins and followed by MS profiling) can provide direct and intrinsic information regarding the proteins and allow for the identification and quantification of all proteins contained in a sample.46 However, long-term trypsin-based proteolysis is still a bottleneck in high-throughput proteomics. We then apply the designed GST nanoreactor for remote modulation of trypsin bioactivity and high efficient proteolysis in proteomics. We first used BSA as a model protein to explore and optimize the GST nanoreactor for ultrafast and remote proteolysis. The trypsin density in GST nanoreactor and the amount of GST nanoreactor were studied and optimized. As shown in Figure S5A, the increase of trypsin density contributes to the effective digestion and protein identification due to the introduction of more active sites; however, overloading of trypsin in the GST nanoreactor would lead to deteriorated digestion performance, because too much trypsin would block the nanochannels and hinder the access for substrate proteins. Apparently, increasing the ratio of GST nanoreactor to the substrate would enhance proteolysis efficiency, when the amount of the GST nanoreactor is less than 25 µg (Figure S5B). Further increasing the amount of GST nanoreactor would result in deterioration of proteolysis, because overdoes of GST nanoreactor with incorporated graphene would lead to the overheating. As a NIR light-responsive nanoreactor, the bioactivity of GST nanoreactor can be controlled by either laser power or exposure time (Figure 4A, B). More importantly, the stimulus-responsive feature of the GST nanoreactor enables us to modulate the bioactivity on-demand by simply


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switching the laser on and off (Figure 4C). Figure 4D displays the mass spectrum (MS) of BSA digested by the GST nanoreactor with NIR radiation in 1 minute. Surprisingly, assisted by the GST nanoreactor upon NIR radiation, successful profiling proteins could be achieved in seconds. High-quality MS with high peak intensity and signal-to-noise (S/N) ratio was observed, and peptide identification with high confidence was obtained (Table S1). However, without the NIR light radiation, the proteolysis efficiency of GST nanoreactor was dramatically decreased (Figure 4E), revealing the NIR light radiation-induced in situ heat is critical to boost the proteolysis efficiency of GST nanoreactor. As a control, digestion of the substrate protein using free trypsin by heating in 1 minute is compromised (Figure S6), although extending the digestion time to overnight would lead to effective digestion of the substrate protein (Figure 4F). Apparently, reduction of protein digestion time from overnight to several seconds by the assistance of GST nanoreactor can significantly improve the speed and throughput for practical application in proteomics.



Figure 4. Remote modulation of GST nanoreactor. Control the proteolysis efficiency of GST nanoreactor by (A) laser power and (B) exposure time. (C) Remote modulation of proteolysis by switching NIR laser On/Off. Mass spectra of the model protein BSA proteolysis using different strategies: (D) GST nanoreactor with NIR laser on for 1 minute; (E) GST nanoreactor under with NIR laser off for 1 minute; (F) Overnight proteolysis using pure trypsin. To study the mechanism of the light-enhanced proteolytic efficiency, we tested the proteolytic activity of PDA modified GS nanostructure, trypsin, and the mixture of GS and trypsin under NIR radiation. Typically, the proteolytic activity of trypsin is sensitive to temperature (Figure S7). However, the systemic temperature has no significant influence on the proteolysis of substrate proteins by the GST nanoreactor under NIR radiation (Figure S8A). It can be attributed to the NIR light radiation-induced in situ heating that promotes the proteolytic activity of trypsin


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in the GST nanoreactor. Notably, under NIR radiation, PDA modified GS nanostructure cannot effectively digest the substrate proteins (Figure S8B) due to the absence of specific cleave sites from trypsin. What’s more, in comparison to GST nanoreactor, the proteolytic activity of the free trypsin under NIR radiation is limited (Figure S8C), indicating the important role of GST nanoreactor. Furthermore, the proteolytic activity of the mixture of trypsin and GS nanostructure under radiation of NIR is also compromised (Figure S8D), which could be ascribed to the lack of immobilization of trypsin in the GS nanostructure. The substrate proteins would dynamically exchange trypsin absorbed on the surface of GS nanostructure, which will lead to the low proteolytic efficiency. However, for GST nanoreactor, the trypsin molecules are separately assembled in the nanocompartment in GST nanoreactor and the in situ ratio of trypsin to the substrate is much higher than the free trypsin in solution. More importantly, when trypsin is immobilized in the GS nanostructures, the trypsin structure is more stable than that in solution and more active sites could be exposed, providing more chance to interact with the substrate proteins. Besides, the unique hierarchical porous structure of GST nanoreactor would also contribute the rapid absorption of substrate proteins through the large nanopores,47, 48 while the digested products can be diffused out of the GST nanoreactor rapidly through the nanopores. Moreover, the capability of NIR laser to modulate and/or promote biological process has been well documented at both cell and tissue level in biomedical field.49, 50 Therefore, the synergistic effect of the GS nanostructure, the trypsin immobilization, and the laser radiation is responsible to achieve high efficiency in NIR induced proteolysis. GST nanoreactor is characterized by its nanocompartment structure, and the trypsin molecules are covalently assembled in the nanocompartment with proper molecule density. Thus, it is easy to recover the GST nanoreactor from the digestion products by centrifugation, which is not



possible for free trypsin molecules. As evaluated using the FITC-trypsin model, leakage of trypsin from GST nanoreactor is negligible during the treatment (Figure S9). On the other hand, the designed nanoreactor not only greatly stabilizes the trypsin structure but also avoids possible self-digestion arising from the proximity between individual trypsin molecules, although trypsin, as an active protein, is easy to lose the activity due to its poor thermal stability and selfdegradation. The stability of the GST nanoreactor stored at ambient conditions was evaluated. To our surprise, the proteolytic performance of GST nanoreactor was only slightly decreased with the prolonged room temperature storage up to 30 days (Figure 5A), while the stability of the free trypsin in solution dramatically deteriorated in 5 days. High-performance protein digestions could be achieved over the one-month period, and the identified peptide patterns are similar to those obtained using the fresh GST nanoreactor (Figure 5B). However, for stored pure trypsin, the identified peptide patterns changed drastically, due to the presence of degradation fragments of trypsin and the loss of characteristic peptides from the substrate protein (Figure S10). Tissue microenvironment is critical in various aspects of in vivo biological processes, such as morphogenesis and tissue formation,51 tumorigenesis and metastasis,52 stem cell differentiation,53 immunosurveillance,54 and homeostasis maintenance and regulation, etc.55 Mass spectrometrybased proteomic analysis is an important tool to study tissue microenvironment,56 because it focuses on the study of large-scale proteins expression changes in structure and function via a global perspective.57 Conventionally, proteins are extracted from tissue samples by biopsy or sacrificing the animal, digested into peptides, and profiled by various methods of MS.58 However, tissue sampling is invasive and can only capture a snapshot of the proteome. Tissues are perfused by blood and lymph, proteins and protein fragments can passively or actively enter the circulation.28 Thus, proteolysis of proteins in tissue and detecting those resultant peptides in


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circulation could produce informative traces of tissue microenvironment. To study whether GST nanoreactor could be used for protein modulation and ultrafast proteolysis in deep tissue, pork muscle tissues with different thickness were used as the tissue models plated on the top of the GST nanoreactor (Figure S11). Upon irradiation with NIR light, the temperature of the tissue was only slightly increased, while the temperature of the GST nanoreactor under the tissue was increased rapidly (Figure 5C). Furthermore, the bioactivity of GST nanoreactor reduced with the increase of tissue thickness, but effective proteolysis (∼63%) was still observed even with 5 mm tissue (Figure 5D). These results indicate that the GST nanoreactor could be activated by NIR light in deep tissue, and the protein activity could be remotely modulated by the combination of GST nanoreactor and NIR light radiation.

Figure 5. (A) Stability of the GST nanoreactor and trypsin solution stored at the room temperature over a month. Note: after 5 days, trypsin almost lost its capability for proteolysis and the model protein treated by trypsin stored longer than 5 days cannot be identified by MS. (B)



Peptide patterns of the model proteins digested by GST nanoreactor stored for different days. (C) Illustration and thermal imaging of deep tissue NIR irradiation (laser power: 2W) for 1 minute by covering the sample with pork tissue. (D) Proteolysis by GST nanoreactor under NIR radiation through pork tissue of 0, 2.5, or 5 mm in thickness. To further verify the universal effectiveness of the GST nanoreactor and retained trypsin functionality by NIR light activation, we applied the GST nanoreactor for proteolysis of various proteins including beta-lactoglobulin, myoglobin, and cytochrome c were used. As the conventional strategy of using free trypsin in solution for proteolysis in 1 minute is not possible, we extended the proteolysis time to 12 hours for comparison. Apparently, assisted by NIR radiation, GST nanoreactor can effectively digest the substrate proteins in 1 minute and highquality MS were recorded (Figure S12A, C, and E) which is comparable to those obtained using the conventional long-term (12 hours) in solution proteolysis (Figure S12B, D, and F). Based on the high-quality MS, these proteins were identified with high sequence coverage and peptide match number (Table S2-4). The proteolytic performance of the GST nanoreactor (one-minute proteolysis for the identification of 6 myoglobin peptides with 94% sequence coverage) is superior to the trypsin-loaded porous silica composites (10 to 30 minutes or overnight proteolysis for the identification of 3 to 16 targeted peptides with sequence coverage of 13% to 86%),48, 59, 60. Furthermore, according to the overlap analysis, we observed no significant divergence between the GST nanoreactor and free trypsin in the digestion selectivity (Figure S12G), indicating the trypsin functionality in GST nanoreactor has not been altered. These results demonstrate GST nanoreactor can be used for digestion of wide range of proteins, which is important for proteolysis in practical proteomics.


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Application of GST nanoreactor in Proteomics. Encouraged by above results, a complex sample of protein mixture extracted from whole cell lysate (MDA-MB-231 cells) was used to explore and evaluate the potential application of the GST nanoreactor in practical proteomics. The products generated from the GST nanoreactor were analyzed by MS and then coupled with database search for protein identification. As a comparison, proteolysis using conventional overnight strategy using free trypsin was also conducted. As a result, 745 proteins with high confidence (p600 kDa) were also identified for the sample treated using GST nanoreactor, implying that GST nanoreactor is effective for proteolysis of large proteins. It can be attributed to the unique nanocompartment structure of the GST nanoreactor, which can absorb and confine the large proteins rapidly and thus contribute to the high efficient proteolysis. Furthermore, the broad distribution of their pIs further demonstrates the capability of GST nanoreactor for efficient digestion of wide range of proteins. Although the proteolysis time is dramatically decreased for the sample treated using GST nanoreactor, 432 proteins were identified for samples treated by both approaches while 313 proteins were uniquely identified for samples treated by GST nanoreactor (Figure 6C). At the peptide level, the molecular weights of the identified peptides were mainly concentrated between 1 kDa and 4 kDa, which confirms the proteins were well digested (Figure 6D). What’s more, over 96.7% peptides were detected with expected perfect termini (Figure 6E) while most of the peptides were detected with none or one missed cleavage



(Figure 6F) for the sample treated using GST nanoreactor, which is comparable to those observed from proteins treated by conventional strategy. Apparently, in comparison to conventional strategy of using free trypsin, the ultrafast proteolysis doesn’t sacrifice the protein identification, although the digestion time is decreased from overnight to seconds.

Figure 6. Rapid proteolysis of complex cell lysate using the GST nanoreactor in practical proteomic. (A) Cumulative sequence coverage of the identified proteins using the GST nanoreactor and free trypsin. (B) The Mw and pI distribution of the identified proteins from samples treated with the GST nanoreactor and free trypsin; (C) The crossing analysis of the


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identified proteins from the samples treated with GST nanoreactor and free trypsin. (D) Mw distribution of identified peptides from samples treated with the GST nanoreactor and free trypsin; (E) The specificity analysis of identified peptides from samples treated with the GST nanoreactor (outer ring) and free trypsin (inner ring). (F) Missed cleavage results of identified peptides from samples treated with the GST nanoreactor and free trypsin. Our results have demonstrated the construction of novel GST artificial nanoreactor for remote and controllable modulation of trypsin bioactivity and application in ultrafast proteolysis. Trypsin biomolecules were assembled in the nanocompartments of designed hierarchical graphene-silica nanostructure via a bio-adhesive strategy. Different from the time-consuming strategy of using free trypsin in solution, the GST nanoreactor can achieve ultrafast proteolysis in seconds under remote NIR radiation without scarification of the efficient protein identification. Thus, GST nanoreactor could offer substantial advantages for application in high throughput proteomics and, especially, time-limited proteomics diagnosis. Furthermore, the GST nanoreactor is also characterized by their NIR stimulus responsiveness, which enables the remote and controllable proteolysis. NIR light has high penetration capability in tissue, which makes GST nanoreactor a highly potential and ideal candidate for in vivo application. Thus, the GST nanoreactor provides a potential tool for expanding proteomics in vivo. In addition, bioactivity of the GST nanoreactor was retained for nearly one month, owing to the unique nanocompartment structure. This also addresses a current challenge of biomolecule protection. Notably, other biomolecules can also be assembled in the rational designed GS hybrids. Therefore, the proposed strategy can be extended to modulate a wide range of bimolecular activity or construct of multifunctional nanosystem for biomolecule protection and delivery in biomedical diagnosis or therapy.



4. CONCLUSION In conclusion, we have designed and constructed the novel GST artificial nanoreactor for modulating the bioactivity of trypsin molecules by NIR light. Trypsin biomolecules are spatially confined and protected in the hierarchical nanocompartment architecture, and the bioactivity can be remotely controlled upon NIR radiation due to the unique photothermal effect of GST nanoreactor. We successfully demonstrated reliable proteolysis by the GST nanoreactor in seconds, which is far superior to the slow proteolysis using free trypsin in solution. Furthermore, the unique stability of the GST nanoreactor enables the excellent utilization and persistence. We envision this work not only provides a promising tool of ultrafast and remotely controllable proteolysis for high throughput proteomics and even in vivo proteomics in disease diagnosis and other biomedical applications but also paves the way for exploring smart artificial nanoreactor in bimolecular modulation to gain insight in dynamic biological transformation. ASSOCIATED CONTENT Supporting Information. Characterization of GST nanoreactor and detailed study and optimization NIR modulation of bioactivity of GST nanoreactor. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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In Situ Caging of Biomolecules in Graphene Hybrids for Light

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