Journal of Chromatography A, 1388 (2015) 158–166

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Investigation of bi-enzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics Lulu Shangguan a,1 , Lingyi Zhang a,∗,1 , Zhichao Xiong a , Jun Ren a , Runsheng Zhang b , Fangyuan Gao a , Weibing Zhang a a

Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China Shanghai Key Laboratory of Crime Scene Evidence, Shanghai Research Institute of Criminal Science and Technology, Shanghai Public Security Bureau, Shanghai, P.R. China b

a r t i c l e

i n f o

Article history: Received 19 December 2014 Received in revised form 11 February 2015 Accepted 12 February 2015 Available online 17 February 2015 Keywords: Nanoparticle Hybrid organic–inorganic monolith Bi-enzymatic reactor Proteolysis Membrane protein

a b s t r a c t The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bienzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic–inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24 h to 50 s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention, due to the high accuracy, reliability, and reproducibility in protein identification [1,2]. In this strategy, protein samples are firstly digested to peptides by a protease (typically trypsin), followed by LC-MS analysis for peptide and protein identification. Thus, how to realize rapid and efficient generation of peptides becomes one of the most challenging steps for such MS-based proteomics analysis. The challenges are particularly stringent for membrane proteins as they demonstrate poor solubility, low abundance and relative paucity of

∗ Corresponding author. Tel.: +86 021 64253977; fax: +86 021 64233161. E-mail address: [email protected] (L. Zhang). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.chroma.2015.02.040 0021-9673/© 2015 Elsevier B.V. All rights reserved.

tryptic cleavage sites [3], which make them notoriously difficult to study and consistently underrepresented in proteomic analyses [4]. It has been shown that the use of multiple proteases can greatly increase the sequence coverage of identified proteins by effectively maximizing number of generated peptides for MS identification in large-scale proteomic analysis [5,6]. This versatile approach has made some success in solution-based tandem digestion. Glatter et al. [7] accomplished large-scale quantitative assessment of proteins by using different in-solution digestion protocols; the results revealed that superior cleavage efficiency of tandem Lys-C/trypsin proteolysis was over trypsin digestion. Wisniewski et al. [8] also performed consecutive proteolytic digestion with Lys C and trypsin by an enzyme reactor. The tandem use of endoproteinases enabled identification of up to 40% more proteins and phosphorylation sites in comparison to the commonly used one-step tryptic digestion. However, solution-based tandem digestion strategy not only has the conventional solution digestion drawbacks as autolysis, poor enzyme to substrate ratio, but also extends digestion time up to 24 h

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or more which greatly limits the sample processing throughput. Therefore, the application of enzymatic reactor with multiple proteases immobilized may combine the advantages of immobilized protease and multiple protease digestion to achieve fast, highly efficient and complete digestion for complex protein samples. Due to the advantages of low enzyme autolysis, high enzyme-tosubstrate ratio, time-saving and reusability, immobilized enzyme reactor (IMER) has drawn much attention recently [9–11]. For IMER, enzymes can be immobilized by several mechanisms: sol–gel trapping [10], layer-by-layer assembly [12], cross-linking [13], covalently bonding or physical adsorption [14,15]. And variety of materials have been used to support enzymes, including polymer membranes [16], microchips [17], capillary columns [18], micro/nanoparticles [19] and monolith materials [20]. Among various matrices, monolithic materials are used more widely owing to its obvious advantages such as easy fabrication and modification, good biological compatibility [21]. Combining the advantages of organic and inorganic monoliths, the organic–inorganic hybrid monoliths attract much attention due to the characteristics of better pH stability and less shrinkage [22]. In addition, with large surface area and some special surface properties [23], nanoparticle also shows great potential as excellent enzyme support [24,25]. In our recent work, we designed a novel IMER by immobilizing trypsin on a composite support of hybrid organic–inorganic monolith with SBA-15 nanoparticles embedded in silica capillary. Owing to higher trypsin immobilization amount, the IMER was successfully applied to digestion of standard proteins and rat liver proteins with high sequence coverages, while the digestion time was reduced to only 19 s in dynamic mode [26]. In general, only one protease was immobilized (typically trypsin) in IMER [17,27,28], which limits digestion efficiency and tends to generate missed cleaved peptides, especially for proteins lacking of lysine (R) and arginine (K) residues. Besides trypsin, other endoproteases such as chymotrypsin [16,29], Glu-C [30,31], Lys-C [32] can also be used for MS-based protein analysis to cover the complete sequence of proteins. The specific enzymolysis sites of chymotrypsin are phenylalanine (F), tryptophane (W), tyrosine (Y), and leucine (L), methionine (M) are less specific enzymolysis sites for it [33–35]. Considering the completely orthogonal specificities of these two proteases, the combination of trypsin and chymotrypsin may lead to better digestion. Fischer et al. [33] had used mixed enzymes of trypsin and chmotrypsin for hydrolysis of membrane proteins in an organic solvent; the combination significantly improved the identification of hydrophobic peptides with distinctly higher sequence coverage of transmembrane regions. Zhou et al. [16] proposed a novel enzymatic reactor by simultaneously immobilizing trypsin and chymotrypsin on biocompatible PVDF membranes, and this bi-enzymatic reactor could produce enhanced rapid digestion of standardized prototypic proteins, hydrophilic proteins and hydrophobic transmenbrane proteins. Temporini et al. [29] also proposed a multi-enzymatic approach by synchronously bonding trypsin and chymotrypsin to an monolithic silica column in a one-step reaction via epoxy-groups, which showed good digestion efficiency and increased the confidence degree in proteomic analysis. In this paper, a bi-enzymatic reactor based on monolith with nanoparticles embedded was prepared to achieve complementary digestion of complex protein samples. The monolith and nanoparticles were modified with trypsin and chymotrypsin by using covalent bonding with glutaraldehyde as bridging reagent and chelation with copper ions, respectively. The preparation and operation conditions including nanoparticles percentage, the inner diameter of silica capillary and the length of the bioreactor were optimized. Carbonic anhydrase was used to evaluate the performance of the bi-enzymatic reactor. The advantages and proteolysis characteristics of the bi-enzymatic reactor were further demonstrated by its

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application in membrane proteins from rat liver and proteins of rat liver extract by comparing with two single protease reactors and the solution-based tandem digestion using free proteases. 2. Materials and methods 2.1. Instruments A syringe pump (Baoding Longer Precision Pump Co., Ltd., China) was used to push the sample solution through capillary reactor. Sol–gel solution and samples were mixed by a vortex vibrator (QL901, Kylin–Bell Lab Instruments Co., Ltd., China). The nanoparticles were homogeneously dispersed in solution by an ultrasonic cleaner (SZ-80, Hangzhou Sagee Instrument Co., Ltd., China). The temperature of digestion was controlled by a column oven (Dalian Elite Analytical Instruments Co., Ltd., China). 2.2. Reagents and materials Carbonic anhydrase (bovine erythrocytes), BSA (bovine serum), trypsin (bovine pancreas), ␣-chymotrypsin (bovine pancreas), dithiothreitol (DTT) and iodine acetamide (IAA) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Tetraethoxysilane (TEOS, 98%), 3-amino-propyltriethoxysilane (APTES, 99%) and tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl, 99%) were purchased from ACROS (Shanghai, China). Cetyltrimethyl ammonium bromide (CTAB) was purchased from Crystal Pure Reagent (Shanghai, China). 3-Glycidoxypropyltrimethoxysilane (GLYMO), iminodiacetic acid (IDA), benzamidine hydrochloride, sodium cyanoborohydride (NaBH3 CN) and urea were purchased from Aladdin (Shanghai, China). Formic acid (FA) was from Fluka (Buches, Germany). HPLC-grade acetonitrile (ACN) was from Merck (Darmstadt, Germany). Fused silica capillary with diameters of 75 and 100 ␮m i.d. was obtained from Sina Sumtech Co., Ltd. (Hebei, ˚ were purchased from China). C18 AQ beads (3 and 5 ␮m, 120 A) Daiso (Osaka, Japan). 2.3. Preparation and modification of nanoparticles The SBA-15 nanoparticles were prepared as previous report [36]. The modification procedure of SBA-15 nanoparticles with carboxylic group is shown in Fig. 1A. Firstly, the iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane (GLYMO-IDAsilane) was synthesized according to reference [37]. The SBA-15 nanoparticles (0.01 mg) were dispersed homogeneously in 25 mL of ethanol by vortexing and sonicating, and 5 mL of GLYMO-IDAsilane (pH 6.0) was added into the suspension. Then the mixture was incubated in a water bath at 40 ◦ C for 24 h. Finally, the SBA-15 nanoparticles were cleaned with ethanol and then put into oven at 60 ◦ C overnight to get IDA modified nanoparticles (SBA-15-COOH). 2.4. Preparation of hybrid organic–inorganic monolith with SBA-15 nanoparticles embedded The hybrid organic–inorganic monolithic column was prepared as reported in our previous work [38]. TEOS (112 ␮L), APTES (118 ␮L), ethanol (215 ␮L), water (32 ␮L) and CTAB (8 mg) were mixed and sonicated to obtain a homogeneous solution (donated as MPS, monolith preparation solution). Then, 100 ␮L of MPS was mixed with 2.0 mg SBA-15 nanoparticles, followed by vortex shaking and sonication in turn 1 min for each to form a homogeneous solution at 0 ◦ C, which was filled into the pretreated capillary with appropriate length by a syringe. After both ends being sealed, the capillary was put into a water bath at 40 ◦ C for 24 h. Subsequently, the obtained hybrid monolithic capillary column was flushed with

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Fig. 1. Preparation of bi-enzymatic reactor. (A) The carboxyl modification of SBA-15 nanoparticles. (B) Enzyme immobilization on the hybrid monolithic support. (C) Enzyme immobilization on the nanoparticle support.

ethanol, water and 100 mmol/L NaH2 PO4 (pH 8.0) to remove the unreacted residuals, respectively.

2.5. Preparation of bi-enzymatic reactor with trypsin and chymotrypsin immobilized Two different enzymes were immobilized to monolithic support and nanopartilces, respectively. The immobilization of enzyme on the hybrid monolith support was described in our previous report [26]. The hybrid monolithic capillary was rinsed with 100 mmol/L phosphate buffer (pH 8.0) containing 10% (v/v) glutaraldehyde for 6 h, and then 100 mmol/L phosphate buffer for 30 min at room temperature. After that, trypsin was immobilized onto the monolithic support by continuously pumping 2 mg/mL trypsin dissolved in 100 mmol/L phosphate buffer (pH 8.0) containing 0.05 mol/L benzamidine and 5 mg/mL NaBH3 CN into the capillary at 4 ◦ C for 24 h. Subsequently, the non-adsorbed enzyme was removed by purging the capillary with 100 mmol/L phosphate buffer (pH 8.0) containing 20% (v/v) ACN for 4 h. Then the monolith was washed with 1 mol/L Tris-HCl (pH 8.0) for 4 h to quench the unreacted aldehyde groups. The immobilizing procedure of trypsin is presented in Fig. 1B. The immobilization of another enzyme on the nanoparticles was similar to the previous report [39]. The enzymatic reactor with trypsin immobilized was rinsed with water for 30 min, and then 50 mmol/L CuSO4 solution was continuously pumped into the reactor for 1 h, followed by washing with water to remove the un-chelated Cu2+ . After that, chymotrypsin was immobilized onto the nanoparticles by introducing 2 mg/mL chymotrypsin dissolved in 100 mmol/L phosphate buffer (pH 8.0) containing 0.05 mol/L

benzamidine into the capillary at 4 ◦ C for 2 h. Then the nonspecifically absorbed chymotrypsin was flushed out by pumping 50 mmol/L NH4 HCO3 buffer (pH 8.0) containing 20% (v/v) ACN for 2 h at 4 ◦ C. Thus, the hybrid organic–inorganic monolithic bi-enzymetic reactor with SBA-15 nanoparticles embedded was obtained and stored in 50 mmol/L Tris-HCl buffer (pH 7.5) containing 10 mmol/L CaCl2 and 0.02% NaN3 (w/v) at 4 ◦ C before usage. The immobilization procedure of enzyme on the nanoparticles is shown in Fig. 1C. This bi-enzymatic reactor was denoted as MTSC. For comparison, with the same preparation conditions, another bi-enzymatic reactor (denoted as MCST) was prepared, in which trypsin was immobilized onto the nanoparticles and chymotrypsin was immobilized onto the monolith. In addition, the enzymatic reactors with single enzyme (trypsin or chymotrypsin) immobilized on the dual matrixes were prepared with the same two-step immobilization procedure.

2.6. Determination of immobilized enzyme The amount of immobilized enzyme on microreactor was determined by Bradford assay by spectrophotometer, which is similar to the previous report [42]. Briefly, the microreactor was chopped into small pieces with a length of 2 cm and then 200 ␮L 100 mmol/L NaOH was introduced into the reactor with a constant flow rate of 1 ␮L/min at room temperature to cleave enzyme completely. Then the collection was centrifuged at 12,000 r/min for 15 min and 150 ␮L of the supernatant was collected and diluted by 850 ␮L 100 mmol/L NaOH for further analysis. Enzyme standard solutions were prepared in 100 mmol/L NaOH in the concentration

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range of 1–10 ␮g/mL. One milliliter of each enzyme standard and the cleaved enzyme solution were mixed with 1 mL of Bradford reagent, respectively. After each mixture was incubated at room temperature for 5 min, the absorbance was measured with a spectrophotometer at 595 nm, and the immobilized enzyme content was calculated. 2.7. Extraction of rat liver proteins The rat liver was dissected, cut into small pieces, and cleaned with cold saline (0.9% NaCl). Then it was homogenized in buffer containing 2 mol/L NaCl, 50 mmol/L phosphate buffer (PBS) (pH 7.4) and 1% (v/v) protease inhibitor cocktail at ice bath. The homogenate was centrifuged at 1600 × g at 4 ◦ C for 1 h. The supernatant was collected and stored at −80 ◦ C till further analysis. 2.8. Extraction of rat liver membrane proteins The rat livers were dissected, cut into small pieces and subjected to the cellCribble, then the collection was centrifuged at 1000 × g at 4 ◦ C for 5 min. The precipitate was cleaned with cold 50 mmol/L phosphate buffered saline (PBS) (pH 7.4) at ratio of 1:10 (v/v) for two times, then centrifuged at 1000 × g at 4 ◦ C for 5 min. The supernatant was resuspended in hypotonic buffer containing 1% (v/v) protease inhibitor cocktail and put into ice bath for 10 min, then centrifuged at 1000 × g at 4 ◦ C for 10 min. The precipitate was resuspended in hypotonic buffer containing protease inhibitor cocktail at 4 ◦ C for 10 times, then centrifuged at 2500 × g for 5 min. The supernatant was centrifuged at 10,000 × g at 4 ◦ C for 60 min, and then the precipitate was collected and resuspended in 50 mmol/L ammonium bicarbonate containing 8 mol/L urea in ice bath, then sonicated for 1 s repeatedly with 2 s interval, till the solution was clear. Finally, the solution was centrifuged at 14,000 × g at 4 ◦ C for 15 min, then the supernatant was collected, quantified and then stored at −80 ◦ C till further analysis. 2.9. Protein digestion by solution-based free proteases or immobilized enzymatic reactor The protein was dissolved in 50 mmol/L NH4 HCO3 (pH 8.0) containing 8 mol/L urea for denaturation followed by DTT reduction and IAA alkylation. And the denatured sample was diluted by 50 mmol/L NH4 HCO3 (pH 8.0) to needed concentration before using. For solution digestion, trypsin was introduced into the denatured protein solution at protein-to-enzyme ratio of 50/1 (w/w), and the mixture was incubated at 37 ◦ C for 12 h, and then cooled to room temperature. For further digestion of trypsin generated peptides by chymotrypsin, chymotrypsin was added to the trypsin digestion with a protein-to-enzyme ratio of 50/1 (w/w), and the digestion was performed at 37 ◦ C for 12 h. Finally, formic acid was added to the solution to terminate the reaction. For the immobilized enzymatic reactor digestion, the denatured sample was introduced into the reactor with a constant flow rate of 0.5 ␮L/min at 37 ◦ C. The digestion products were collected and then stored at −20 ◦ C for further analysis. 2.10. MALDI-TOF MS analysis All MALDI-TOF MS analyses were performed on an AB Sciex 4800 MALDI-TOF/TOF mass spectrometer (AB Sciex, USA) equipped with a 200 Hz repetition rate and wavelength of 355 nm in reflex positive ion mode. 0.5 ␮L of the digestion products and 0.5 ␮L of 10 mg/mL cyano-hydroxycinnamic aicd (CHCA) matrix solution dissolved in aqueous solution containing 50% (v/v) ACN and 0.1% (v/v) TFA were spotted on the MALDI plate and dried at room temperature prior to MALDI-TOF MS analysis. And the data were searched by Mascot

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as engine (Matrix Science, London, UK) and Swissprot to identify protein based on peptide mass spectra. 2.11. Nano-LC/MS/MS analysis The digestion products of carbonic anhydrase were analyzed by a LTQ mass spectrometer (Thermo, San Jose, CA) with a nanospray source; the peptides from rat liver proteins were analyzed by a LTQ Orbitrap mass spectrometer (Thermo, San Jose, CA) with the same nanospray source; the peptides from membrane proteins of rat liver were analyzed by a LTQ Orbitrap Velos (Thermo, San Jose, CA) with an Accela 600 HPLC system (Thermo, San Jose, CA) for separation. A capillary column (75 ␮m i.d. × 15 cm) was firstly manually pulled to a fine point as spray tip and then packed with C18 AQ beads ˚ Michrom Bio Resources). Formic acid (0.1% v/v) in (5 ␮m, 120 A, water and formic acid (0.1% v/v) in acetonitrile were applied as the mobile phase. Gradient elutions from 5% to 35% (v/v) of the 0.1% (v/v) formic acid in acetonitrile in 90 min for carbonic anhydrase, 120 min for rat liver proteins and 150 min for membrane proteins were performed to elute each sample in one-dimensional RP LC/MS/MS. All MS and MS/MS spectra were acquired in datadependent mode with the 7 (for LTQ MS) or 10 (for LTQ Orbitrap and LTQ Orbitrap Velos MS) most intense ions fragmented by collisioninduced dissociation (CID). 2.12. Protein identification Raw files acquired from the LTQ MS were searched against carbonic anhydrase protein sequence from a uniprot database of bovine with mascot (Matrix Science, version 2.3.0). Peptides were searched with fully tryptic cleavage constraints, and up to two missed cleavage sites were allowed. Cysteine carbamidomethylation (+57.0215 Da) was set as a static modification, and methionine oxidation (+15.9949 Da) was set as a variable modification. Peptide mass tolerance was set to 2 Da and fragment mass tolerance was set to 0.8 Da. The peptide and protein false discovery rates (FDRs) are from the internal Mascot decoy database search function. Peptides with FDR < 1% were accepted for identification. Raw files from the LTQ Orbitrap MS and LTQ Orbitrap Velos MS were searched with MaxQuant (version 1.3.0.5) against a UNIPROT database of mouse (ftp.uniprot.org). In the trypsin digestion, peptides were searched with fully tryptic cleavage constraints and up to three missed cleavage sites were allowed; in the chymotrypsin or multiple proteases (trypsin + chymotrypsin) digestion, up to seven missed cleavage sites were allowed. Cysteine carbamidomethylation (+57.0215 Da) was set as a static modification, and methionine oxidation (+15.9949 Da) was set as a variable modification. Peptide mass tolerance was set to 10 ppm and fragment mass tolerance was set to 0.5 Da. FDRs limit was set to 0.01. The other settings were the same as the conventional search. 3. Results and discussion 3.1. Selection of enzymes and immobilization methods In this work, we proposed a novel bi-enzymatic reactor by immobilizing two complementary enzymes by cross-linking and metal chelating onto monolithic support and nanoparticles, respectively. Trypsin and chymotrypsin were employed to prepare bi-enzymatic reactor, as the proteolysis conditions of the two proteases are coincided with each other. They are both active at pH 8.0 and 37 ◦ C [40]. In addition, the specificities of these two enzymes are greatly orthogonal. As shown in Table 1, comparing the digestions of membrane proteins from rat liver by tryptic and chymotryptic reactors, it was worthy noted that only 182 identified proteins were overlapped and the identified peptides were

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Table 1 Numbers of the identified proteins and peptides obtained by tryptic reactor, chymotryptic reactor, MTSC and solution-based tandem digestion (trypsin + chymotrypsin). Identified protein or peptide

Trypsin reactor

Chymotrypsin reactor

Overlap

Sum

MTSC

In-solution

Peptides Proteins

1184 452

1010 336

0 182

2194 606

2891 620

2037 570

Digestion conditions: sample, rat liver membrane proteins ∼1 mg/mL; enzymatic reactor: 100 ␮m i.d. × 5 cm; flow rate: 0.5 ␮L/min; solution-based tandem digestion: enzyme/protein = 1/50 (w/w); digesting time, 12 h for each protease; temperature, 37 ◦ C.

entirely different, indicating completely orthogonal specificity of trypsin and chymotrypsin. Besides, the immobilization mechanisms of covalent bonding with glutaraldehyde as bridging reagent and chelating via Cu2+ are both mild which help to maintain the activity of enzymes [13,41,42]. Moreover, the enzyme immobilized by chelation via copper ion onto the nanoparticles can be easily regenerated by removing Cu2+ and enzymes with 50 mmol/L EDTA solution and re-chelating Cu2+ and immobilizing fresh enzyme onto the matrix. These two proteases do not interfere with each other in digestion procedure because they could be successfully immobilized on the monolith and nanoparticle supports respectively. At the end of the first immobilization procedure, Tris-HCl was pumped into the capillary columns to react with the residual aldehyde groups on the hybrid silica monolith, thus the second enzyme could be immobilized onto the nanoparticles only. Benzamidine dissolved in the immobilization solution could inhibit the first immobilized enzyme from digesting the second enzyme during the second immobilization step, and it also minimized enzyme autoprolysis. Additionally, there is no competition or exchange of the two enzymes, because the C N bonds generated in reaction between glutaraldehyde and enzyme are reduced to C N bond. This procedure is irreversible to avoid the competition or exchange of the two enzymes during the immobilization of the second enzyme. In this work, two different bi-enzymatic reactors were prepared. For MTSC, trypsin was immobilized onto the hybrid monolith support and chymotrypsin was immobilized onto the SBA-15 nanoparticles, and MCST was prepared on opposite. Due to limit of amount of nanoparticles embedded into the monolith, the immobilization amount of enzyme on the nanoparticles was smaller than that on the monolith [26]. Therefore, the relative amount of the two enzymes in the two different bi-enzymatic reactors would be different, which might result in different digestion performance. Based on determination of Bradford assay, 1.85 ␮g of trypsin and 1.21 ␮g of chymotrypsin can be immobilized onto monolithic matrix (1 cm of hybrid monolith with SBA-15 incorporated in 100 ␮m capillary) by covalent bonding with glutaraldehyde as bridging reagent and 0.27 ␮g of trypsin and 0.31 ␮g of chymotrypsin can be immobilized onto SBA-15 particles (1 cm of hybrid monolith with SBA-15 incorporated in 100 ␮m capillary). It is noteworthy that the real immobilization amounts of two enzymes in MTSC and MCST cannot be measured directly because there is no way to remove two enzymes separately. The presence of the first enzyme may influence the immobilization amount of the second one. Since lysine (R) and arginine (K) residues, which are cleavage sites of trypsin are well distributed throughout most proteins [43], trypsin immobilized on monolithic support may play a main role in digestion and chymotrypsin immobilized on nanoparticles may play an auxiliary role to help get higher digestion efficiency for complex samples. 3.2. Characterization of the matrix materials The SBA-15-COOH nanoparticles were characterized by small angle XRD pattern, TEM and N2 adsorption/desorption analysis (see Figs. S1–S3 and Table S1 in supplementary material). From SAXD pattern, three well-resolved peaks in the range of 2 = 0.5–2◦ (one strong peak and two shoulder peaks) can be indexed as the

(100), (110) and (220) reflections of the 2D hexagonal space group (p6mm) which is consistent with description of SBA-15 particles in previous report [36]. TEM image of SBA-15-COOH nanoparticles shows that the rod-like nanoparticles have average length of 2.5 ␮m and diameter of 500 nm. Nitrogen adsorption isotherm of SBA-15-COOH features hysteresis loops with sharp adsorption and desorption branches. The sharpness of the adsorption branches is indicative of a narrow mesopore size distribution and the adsorption average pore diameter is 7.3 nm. The mesopores lead the SBA-15-COOH to have a large surface area 413.2 m2 g−1 and a suitable channel for the enzymes to be immobilized in. The SEM images of the monolith with nanoparticles incorporated (before and after immobilization of enzyme) suggest that the hybrid matrix maintains the porous character of monolithic matrix, and porous structure of monolith still exists after immobilized enzymes. Although some of the mesopores of nanoparticle might be filled with the monolithic material during the synthetic procedure, the total specific area of hybrid material with nanoparticles incorporated was still significantly increased compared with monolith without SBA-15 embedded [44]. Additionally, compared with the amount of enzyme only immobilized on the monolith, the increment of amount of immobilized enzyme on both monolith and nanoparticles indicated that the nanoparticles play an important role in hydrolysis, leading to higher digestion efficiency. 3.3. Optimization of IMER with chymotrypsin immobilized on nanoparticles The preparation conditions of immobilizing trypsin onto the hybrid monolith with nanoparticles incorportated was optimized in our previous work [26]. Before preparing the bi-enzymatic reactor, preparation conditions for immobilization of chymotrypsin onto nanoparticles support were investigated, including nanoparticle concentration, inner diameter of capillary and the length of IMER. The chymotrypsin was only immobilized onto SBA-15 particles by chelation with Cu2+ . All enzymatic reactors were evaluated by digesting 1 mg/mL BSA, and the peptides were analyzed by MALDI-TOF MS. Enzymatic reactors were prepared with different nanoparticle concentrations of 10 mg/mL, 20 mg/mL, 30 mg/mL, and 40 mg/mL in Table 2 Optimization of IMER with chymotrypsin immobilized on nanoparticles. Items

Conditions

Nanoparticle percentage (mg/mL)a Peptide matched Sequence coverage (%) Inner diameter (␮m)b Peptide matched Sequence coverage (%) Length (cm)c Peptide matched Sequence coverage (%)

10 7 9 50 13 36 2 3 5

20 28 42 100 28 42 5 28 42

30 14 20 200 7 8 10 11 14

40 9 8 530 – – – – –

Digestion conditions: sample, 1 mg/mL BSA; flowrate, 0.5 ␮L/min; temperature, 37 ◦ C. a 100 ␮m i.d. × 5 cm. b 20 mg/mL nanoparticle; 5 cm length. c 20 mg/mL nanoparticle; 100 ␮m i.d.

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Table 3 Reproducibility of bi-enzymatic reactor. Items

1

2

3

4

5

Run-to-run

Peptide matched Sequence coverage (%) RSD (n = 5)

54 88.5

54 87.3

54 85.0 2.46%

52 83.8

56 88.5

Batch-to-batch

Peptide matched Sequence coverage (%) RSD (n = 3)

42 85.8

45 86.9 2.83%

39 82.3

– – –

– – –

Digestion conditions: bi-enzymatic reactor, MTSC, 100 ␮m i.d. × 5 cm; sample, 1 mg/mL carbonic anhydrase; flow rate, 0.5 ␮L/min; temperature, 37 ◦ C.

capillary with different inner diameters of 50 ␮m, 100 ␮m, 200 ␮m, and 530 ␮m and different lengths of 2 cm, 5 cm, and 10 cm, respectively. From Table 2, it could be seen that the reactor achieved the highest digestion efficiency with 20 mg/mL of nanoparticles, 100 ␮m i.d. and 5 cm of capillary. The typical MALDI-TOF mass spectrum is shown in Fig. S4. Higher nanoparticle concentration resulted in higher specific surface area, which helped to load more chymotrypsin. Besides, the enlargement of surface area [44] could also increase the chance of protein reacting with enzyme. However, the redundant nanoparticles led to bad permeability of columns and uneven distribution of nanoparticles, which reduced the chance of protein reacting with enzyme, thus the sequence coverage of BSA was decreased when the concentration of nanoparticles increased to 30 and 40 mg/mL. The results were consistent with the conclusion of immobilization of trypsin onto both monolith and nanoparticles in our previous work [26]. Compared with the 50 ␮m i.d. reactor, the 100 ␮m i.d. reactor could provide more immobilization sites, which increased the chances of proteins reacting with enzyme, and consequently led to high digestion efficiency. However, when the inner diameter increased to 200 ␮m, the sequence coverage was decreased to 8% (7 peptides matched). It was because oversize of capillary influenced the formation of monolithic matrix and resulted in unstable bone-structure. Especially, when using 530 i.d. capillary, the matrix would be pushed out of the capillary easily. Moreover, the lifetime of reactors prepared with larger capillary was short due to unstable bone-structure. With the increase of capillary length, longer reaction time and

Fig. 2. Veen diagram of the identified proteins of rat liver membrane proteins by using different digestion methods. Digestion conditions: rat liver membrane proteins, 1 mg/mL; enzymatic reactor, 100 ␮m i.d. × 5 cm; flow rate, 0.5 ␮L/min; 37 ◦ C. Solution-based tandem digestion conditions: enzyme/protein = 1/50 (w/w); 12 h for each protease; 37 ◦ C.

more enzymatic sites resulted in more complete digestion and high efficiency. However, when the length was increased to 10 cm, the sequence coverage decreased to 14%. It was probably because that the protein was excessively digested in long length enzymatic reactors and the resulted peptides were too small to be detected by mass detector, because peptides are measured in the range of 600–4000 Da in high-throughput MALDI experiments. 3.4. Reproducibility and stability of immobilized bi-enzymatic reactor The operational reproducibility and stability of the bi-enzymatic reactor were investigated under the optimum conditions. One milligram per milliliter carbonic anhydrase was consecutively digested with the reactor with flow rate of 0.5 ␮L/min at 37 ◦ C, and the IMER was rinsed with 50 mmol/L NH4 HCO3 (pH 8.0) for 20 min between each run. Then the proteolytic products were analyzed by a LTQ mass spectrometer. As shown in Table 3, the sequence coverage of carbonic anhydrase of the fifth run (88.5%) was the same as the first run, indicating the good stability of the bi-enzymatic reactor. And the relative standard deviation (RSD) value of coverage sequence was 2.46% (n = 5). The batch-to-batch reproducibility of the bi-enzymatic reactor was also investigated by identification of carbonic anhydrase, and the RSD was only 2.84% (n = 3), indicating good preparation reproducibility. To further evaluate the stability of the bi-enzymatic reactor, it was stored in 50 mmol/L Tris-HCl buffer containing 10 mmol/L CaCl2 and 0.02% NaN3 (pH 7.5) at 4 ◦ C for 14 days. The sequence coverage of 1 mg/mL carbonic anhydrase was 85.8% (37 peptides matched), which was compatible with the initial value of 82.3% (39 peptides matched).

Fig. 3. Veen diagram of the identified proteins and peptides from rat liver extract by using bi-enzymatic reactors. Digestion conditions: proteins of rat liver extract ∼0.5 mg/mL. Other conditions see Fig. 2.

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Fig. 5. Veen diagram of identified transmembrane proteins of rat liver membrane proteins obtained by using different digestion methods. Digestion conditions see Fig. 2.

Fig. 4. The GRAVY (A) and mass (B) distributions of identified proteins of rat liver membrane proteins by different digestion methods. Digestion conditions see Fig. 2.

3.5. Evaluation of bi-enzymatic reactors To verify the digestion capacity of the bi-enzymatic reactor, membrane proteins from rat liver was digested, followed by a LTQ Orbitrap Velos nano-RPLC-ESI-MS/MS analysis. Two enzymatic reactors with single protease immobilized and solution-based tandem digestion using two free proteases were carried out for comparison. From Table 1 and Fig. 2, the bi-enzymatic reactor (MTSC) showed better results over the two single enzymatic reactors with obviously increased numbers of identified proteins (>30%) and peptides (>∼150%). Moreover, the identified proteins (620) and peptides (2891) obtained by MTSC were even more than the sum of the two enzymatic reactors with single protease immobilized (606, 2194), which demonstrated that the multiple digestion combining trypsin with chymotrypsin could great increase the digestion efficiency for complex samples and the completeness of digestion to produce more MS detectable peptides benefitting MS identification [5,6] by effectively maximizing the enzymolysis sites. More peptides lead to higher sequence coverage of identified proteins and identification of more proteins. Furthermore, when compared with solution-based tandem digestion using two free proteases, MTSC still had great advantages. As showed in Table 1, total 620 proteins and 2891 peptides were positively identified after about 50 s digestion by the MTSC, whereas only 570 proteins and 2037 peptides were recognized by the in-solution digestion lasting for 24 h. Then, MTSC with

excellent digestion efficacy has great potential to be applied in high throughput proteome analysis. Another bi-enzymatic reactor (MCST) was also prepared and both the two bi-enzymatic reactors were evaluated by performing digestion for 0.5 mg/mL proteins from rat liver extract, and the products were analyzed by a LTQ Orbitrap nano-RPLC-ESI-MS/MS. From Fig. 3, it could be seen that totally 636 proteins were identified by MTSC and 634 proteins were identified by MCST. The number of identified proteins and peptides are relatively small due to low sensitivity of LTQ Orbitrap MS compared with LTQ Orbitrap Velos MS used for membrane proteins. The number of overlapped proteins identified by MTSC and MCST were 487. However, more peptides were identified by MTSC, and the overlap of the identified peptides (1322) was low. The different digestion results might be related to the different relative amounts of the two enzymes in these two bi-enzymatic reactors. In MTSC, there were more trypsin immobilized which led to a more complete digestion and generated more peptides. The digestion results of bi-enzymatic reactors were comparable with that of solution-based tandem digestion lasting for 24 h (2337 peptides and 590 proteins) (Table S2). 3.6. Enzymatic characteristics of bi-enzymatic reactors The mass and GRAVY distributions of identified proteins of rat liver membrane proteins were summarized in Fig. 4. It showed that more proteins and peptides can be identified by MS in nearly whole mass and GRAVY ranges using the bi-enzymatic reactor (MTSC) than using others, which demonstrated universality of bienzymatic reactor. Then we analyzed the transmembrane domains of identified membrane proteins by TMHMM Server, v. 2.0. As it showed in Fig. 5, 106 transmembrane proteins were identified by bi-enzymatic reactor which was obviously more than that obtained by single enzymatic reactors (75, 66), and it was also comparable with that obtained by solution-based tandem digestion, showing high digestion efficiency of bi-enzymatic reactor as well. In addition, the characteristics of the two bi-enzymatic reactors were investigated by applying them to digestion of rat liver proteins, as shown in Fig. 6. Though similar numbers of proteins were identified in whole GRAVY and mass ranges by both bi-enzymatic reactors, there were more peptides identified by

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Fig. 6. The GRAVY and mass distributions of identified proteins (A and B) and peptides (C and D) from rat liver extract by using bi-enzymatic reactors. Digestion conditions: see Fig. 3.

MTSC which led to high sequence coverage and better identified accuracy.

4. Conclusion In this work, the bi-enzymatic reactors with trypsin and chymotrypsin immobilized on monolith and nanoparitcle supports respectively were prepared for digestion of complex samples. Compared with single enzymatic reactors, the bi-enzymatic digestion in IMER improved protein and peptide identifications significantly. Compared with solution-based tandem digestion by the same two proteases, the digestion time of bi-enzymatic reactors was shortened from 24 h to 50 s, indicating great application potential in high-throughput proteome analysis.

Acknowledgements We gratefully acknowledge the support of the National Natural Science Foundation for Young Scientists of China (No. 21105027), the Key Research Project from the Ministry of Public Security (201202ZDYJ005), the National Key Scientific Instrument and Equipment Development Project (2012YQ120044) and foundation of Shanghai Research Institute of Criminal Science and Technology.

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