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Journal of Chromatography B, 967 (2014) 13–20

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rn al hom ep age: www.elsev ier .com/ locate /chromb

ovel regenerative large-volume immobilized enzyme reactor:reparation, characterization and application

uihua Ruana,∗, Meiping Weia, Zhengyi Chena, Rihui Sua, Fuyou Dua, Yanjie Zhengb

College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, ChinaShenzhen Academy of Metrology and Quality Inspection, Shenzhen, 518131, China

r t i c l e i n f o

rticle history:eceived 14 February 2014ccepted 7 July 2014vailable online 12 July 2014

eywords:nzyme immobilizationrganic–inorganic hybrid silica particlesegenerative enzyme reactorn-column digestion

a b s t r a c t

A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared withorganic–inorganic hybrid silica particles and applied for fast (10 min) and oriented digestion of protein.At first, a thin enzyme support layer was formed in the bottom of the small column by polymerizationwith �-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol–gelmethod with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 par-ticles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability oflarge-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c(Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) andCyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at101–105, 156–160, 205–209, 212–218, 229–232, 257–263 and 473–451 of the amino sequence of BSAwhen digesting 1 mg/mL BSA. Eight of common peptides were observed during each of the ten runs oflarge-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the –C N– bond by 0.01 M HCl. The sequence coverage of BSA fromregenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides.

accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When theIMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo,ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective diges-tion property of the large-volume IMER indicated that the regenerative IMER could be tentatively usedfor the production of potential bioactive peptides and the study of oriented protein digestion.

. Introduction

Bioactive peptides have been defined as specific protein frag-ents that have a certain positive bio-functionalities and therefore

lay an important role in health promotion and disease riskeduction [1]. ‘Traditional’ bioactive peptides are manufacturedy transgenic, recombinant, or synthetic methods [2]. But thesepproaches are known to be very expensive and thus are pro-ibitive for large scale applications [1]. Hydrolysis of food protein

s a simple and inexpensive method to convert a protein into freemino acids and short chain polypeptides, which are often inactiveithin the sequence of the parent protein and can be released by

nzymatic hydrolysis [3].The enzymatic hydrolysis approach was widely applied for pro-

ucing peptides [4]. The traditional protein digestion was often

∗ Corresponding author. Tel.: +86 773 5896453; fax: +86 773 5896839.E-mail address: guihuaruan@hotmail.com (G. Ruan).

ttp://dx.doi.org/10.1016/j.jchromb.2014.07.008570-0232/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

performed by the in-solution methods [5], however, the tradi-tional in-solution digestion approach has several drawbacks, suchas time-consuming [6], unavoidable enzyme auto-digestion, sam-ple loss or contamination and inconvenience for automation [7].To overcome these drawbacks, some immobilized enzyme reactors(IMERs) have been developed [8], which have many advantagesincluding higher digestion efficiency, shorter digestion time andlower risk for enzyme autolysis than the traditional digestionmethod [9]. More importantly, some IMERs can be readily con-nected with other components directly for on-line digestion [10].Besides, the immobilized enzyme is more resistant to the unfol-ding of enzyme’s native structure that may be caused by heat andpH changes [11].

For IMERs, to select appropriate supports is very importantand challenging tasks for enzyme immobilization, and many sup-

ports, such as microbeads or particles, magnetic nanoparticles,membranes, capillaries or microchannels, monolithic materials,silica materials and hybrids, sol–gel supports and polymers [12],have been succesfully applied. Among them, the organic–inorganic

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ybrid silica materials, which combine the merits of silica withrganic polymer materials, have the advantages of fast and sim-le preparation, good biocompatibility, high permeability andechanical stability, making them superior supports for enzyme

mmobilization [13]. Moreover, micro-enzyme reactors based onhe organic-inorganic hybrid silica materials in capillary with theiameter 20–100 �m have the advantages of relatively facile prepa-ation, fast mass transfer, low backpressure, and high permeabilityor proteomics study, whose purpose is to comprehensively eluci-ate biological processes by systematically analyzing the proteinsxpressed in a cell or tissue [14]. Therefore, the small scale ofmmobilized enzyme reactors or chips have been used to efficientlyroduce a quite amount of peptides that is enough to be iden-ified by mass spectrometry (MS) because of its speed, accuracy,electivity, and sensitivity. But when it comes to bioactive peptideroduction, fast and efficient methods to produce specific cleavagef peptides still are limited and need to further develop.

Although excellent protein digestion capacity of IMERs wasemonstrated, further regeneration of them can hardly be achievedhen the enzymatic activity is decreased. Recently, the metal-ion

helated regenerable IMER has been proposed to solve the problemf regeneration [15,16]. Ma et al. [16] prepared a kind of metal-on chelated IMER supported on organic–inorganic hybrid silica

onolith, and the IMER could be easily regenerated by remov-ng Cu2+ via EDTA followed by trypsin immobilization with freshu2+ introduced. However, the existence of Cu2+ would potentiallyenature the bioactive peptides if applying this type of IMER forioactive peptide production.

In this work, we developed a novel regenerative large-volumeMER on small column for the protein digestion. Organic–inorganicybrid silica particles were used as the support materials fornzyme immobilization. Based on the fact that the –C N–rosslinking with SiO2 particles and trypsin would be easilyydrolyze under acidic conditions [17], the enzyme activity on

MER was easily recovered by reactivating GA and linking trypsinfter the –C N– was broken by HCl. A new regeneration of IMERethod was developed and was successfully used for digestion of

SA and cytochrome c (Cyt-c).

. Experimental

.1. Materials and chemicals

Cytochrome c (Cyt-c, from bovine heart), bovine serumlbumin (BSA, bovine serum, BR), trypsin (pig pancreas,:250), dithiothreitol (DTT,AR), iodoacetamide (IAA) and eth-lene glycol dimethacrylate (EGDMA) were purchased fromladdin Reagent Co. Ltd. (Shanghai, China). Azobisisobutyroni-

rile (AIBN) was obtained from Shanghai Chemical ReagentShanghai, China). Acetonitrile (ACN, CH3CN) was of HPLC graderom Dikma Co., Ltd. (CA, America). All other reagents weref analytical grade, such as tetraethylortosilicate (TEOS), 3-minopropyltriethoxysilane (APTES, Si(OCH3)3(C3H6NH2)), Trishydroxymethyl) aminomethane (NH2C(CH2OH)3), dodecanolCH3(CH2)10CH2OH), Anhydrous ethanol, Urea (H2NCONH2),

ethyacrylic acid (MAA, H2C C(CH3)COOH), glutaraldehyde (GA,5%, w/v, aqueous solution). Water was purified by an arium ® 611ystem (Sartorius, Germany) with resistance ≥18.2 M�/cm.

.2. Preparation and the performance of IMER

.2.1. Preparation of large-volume IMERA thin organic layer at the bottom of 2.5 mL injector (8.9 mm

.d. × 10.2 mm o.d.) was prepared first. The layer with high mechan-cal strength and permeability was prepared according to Huang’s

r. B 967 (2014) 13–20

method [18] but with some modifications. In details, 68 �L MAA,0.4 mL ACN and 1.6 mL dodecanol were mixed and ultrasonicatedfor 30 min, and then 755 �L EGDMA and 0.0144 g AIBN were addedinto the mixed solution and ultrasonicated for another 15 min.300 �L of the solution was injected into an injector whose headwas sealed previously. The polymerization was performed at 60 ◦Cfor 24 h. The resultant organic layer was rinsed with 5 mL of 10%(v/v, formic acid/methanol) solution to remove porogens, and thenflushed with 5 mL of ethanol to remove formic acid/methanolsolution using a peristaltic pump (HL-2D, Shanghai Huxi AnalysisInstrument Factory Co., Ltd).

The organic–inorganic hybrid silica particles were prepared bysol–gel method [19] with minor modifications. After mixture solu-tion of 8.4 mL of TEOS, 8.85 mL of APTES, 16.125 mL of anhydrousethanol and 2.4 mL of water was gently stirred with magnetic stir-ring at room temperature for 12 h, the organic–inorganic hybridsilica was obtained. Then this silica was dried at 90 ◦C for 4 h andthen milled to particles. The performance of p-MAA-EGDMA mono-lith and the organic–inorganic hybrid silica particles immobilizedwith trypsin were characterized with Hitachi S4800 field emissionscanning electron microscope.

Large-volume IMER was prepared by packing GA activated silicaparticles which was then cross-linked with trypsin. Firstly, 0.25 gof silica particles was activated by 2 mL of 0.01% (w/v) GA at roomtemperature for 1 h. The activated silica particles were transferredinto the prepared injector with 5 mL of ethanol. 5 mL of 100 mMphosphate buffer (pH 8.0) was applied to flush the columns usingthe peristaltic pump before the trypsin immobilization. Finally,2 mL of 10 mg/mL trypsin (100 mM phosphate buffer, pH 8.0) wascontinuously pumped through the activated silica particles at thespeed of 0.1 mL/min at room temperature and the residual trypsinwas flushed out by 100 mM phosphate buffer (pH 8.0). This large-volume IMER was stored in 100 mM phosphate buffer (pH 8.0) at−20 ◦C for further use.

In order to investigate the influence of different conditions tothe preparation of IMER, 0.25 g of hybrid silica particles were acti-vated by 2 mL of 0.01%, 0.1%, 0.2%, 0.4% and 0.6% of GA solution,respectively. Then each GA activated silica particles were used toimmobilize 5.0, 10.0 and 15.0 mg/mL trypsin respectively. Finally,all the large-volume IMERs were subjected for the protein diges-tion.

2.2.2. The reusability and regeneration of large-volume IMERTo verify the reusability of large-volume IMER, the IMER was

used repeatedly 10 times for the digestion of BSA at 50 ◦C for10 min. After each digestion completed, the large-volume IMER waswashed with 5 mL of 20% ACN and 100 mM phosphate buffer (pH8.0), respectively.

For the regenerating of IMER, 5 mL of 0.01 M HCl was used toflush the column at room temperature in 15 min, and then the col-umn was washed with 5 mL of phosphate buffer (100 mM, pH 8.0).Finally, after reactivated by 2 mL of 0.1% GA and the 2 mL freshtrypsin (5 mg/mL, 100 mM phosphate buffer, pH 8.0), the hybridsilica particles were used for enzyme immobilization.

2.3. Sample preparation and protein digestion

The preparation of ginkgo was as our previous work [20].Briefly, crude ginkgo powder was extracted from ginkgo nuts withthe method of alkali-solution. The ginkgo albumin solution wasobtained from the process of crude ginkgo powder (5.0 g) dissolvingin water (50 mL), separating at speed of 10,000 r/min and dialysis

(Reviewer 2).

1 mL of BSA (10 mg/mL), Cyt-c (10 mg/mL) and ginkgo albuminsolution were respectively dissolved in 50 mM Tris–HCl (pH 8.1)containing 8 M urea and then reduced via 0.1 mL of 0.1 M DTT for

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0 min at 50 ◦C. After cooling to room temperature, BSA, Cyt-c andinkgo albumin solution were alkylated in the dark in 0.1 mL of.1 M IAA for 15 min at room temperature, followed by the dilu-ion with 8.8 mL of 50 mM Tris–HCl (pH 8.1) to decrease the ureaoncentration to 0.8 M (Reviewer 2).

The on-column digestion was performed by using the peristalticump to pump 1 mL of the pretreated protein sample (treated withDT and IAA) through the IMER at the speed of 0.1 mL/min at 50 ◦C

n a simple incubator device for about 10 min. After the digestionas finished, the enzymatic hydrolysate was flushed out by 1 mL of

0% (v/v) ACN aqueous solution and collected for LC-ESI MS3 identi-cation. Finally, for washing out the residual peptides or protein inhe IMERs, the large-volume of IMERs were rinsed with 5 mL of 20%v/v) ACN aqueous solution and 5 mL of 100 mM phosphate bufferpH 8.0), respectively.

.4. MS analysis

Aeris PEPTIDE XB-C18 column (Phenomenex, 4.6 mm.d. × 250 mm, 3.6 �m i.d. particle size) was used for peptideeparation, with the flow rate of 300 �L/min. The separationondition has been optimized. Water containing 0.1% (v/v) FAbuffer A) and CH3CN (buffer B) were used to generate a 60 minradient, set as follows: 5% B for 1 min, to 40% B in 33 min, to 95%

in 6 min, kept at 95% B for 6 min, to 5% B in 10 min and keptt 5% B for 4 min. 10 �L of sample was injected for LC-ESI MS3

nalysis. The LTQ-Orbitrap (Thermo-Fisher, San Jose, CA, USA)as operated at positive ion mode. The spray voltage was 3.0 kV,

nd the heated capillary temperature was 300 ◦C. The MS wasperated in the data-dependent mode, in which a survey full scanS spectrum (from m/z 100 to 2000) was acquired in the Orbitrapith a resolution of 30,000 at m/z 400. This was then followed byS2 scans of the most abundant ions and the MS3 scans of first,

econd and third most abundant ions from MS2. The resultingragment ions were recorded in the linear ion trap.

.5. Database searching

Protein identifications based on acquired MS3 spectra werearried out using Xcalibur software (version 2.1) and output asaw files. Then the raw files were converted to mzXML filesy X2XML (version 1.3.0.0, free downloaded from http://omics.nl.gov). Finally, database searching was carried out but inputtingzXML files in MassMatrix (version 2.4.2, free downloaded from

ttp://www.massmatrix.net/mm-cgi/downloads.py). The search-ng parameters for BSA and Cyt-c digestion were list in Table 1S andhe searching parameters for ginkgo were list in Table 2S (Reviewer). The mechanism of searching could be explained in details in theublished papers of Xu et al. [21–24].

. Results and discussion

.1. Preparation of regenerative large-volume IMER

Large-volume IMER with a thin layer as stents were prepared byhe thermopolymerization of MAA and EGDMA with dodecanol ashe porogen and AIBN as the initiator, followed by trypsin immobi-ization and finally being regenerated by HCl treated and trypsine-immobilized. All this process was illustrated in Fig. 1S. Aftereing used for several times, the large-volume IMER was treatedith HCl. Undergoing the above process of immobilization again,

he active of IMER recovered but the back pressure of the IMER

ncreased a little bit because of the activation of the silica particles inhe acid condition. After 5 times of regenerating, the back pressuref the IMER became so high that the liquid could not pump throught. This may cause by the dissolving and depositing of silica particles,

. B 967 (2014) 13–20 15

which undergoing the extreme pH transformation from the acid tobase condition during the process of regenerating IMER. It can beseen from Fig. 2S that the size of these particles was nanometerlevel although the particles became agglomerate.

Anyway, the layer is worthy to be highlighted because it playsan important role in building of this large-volume IMER. With thehierarchical mesoporous or macroporous structure of enhancedpermeability, this layer would be an ideal choice for peptides sepa-ration [25] (Viewer 2). It can be seen in Fig. 3S, the pores of thelayer was less than 1 �m. As the silica particles could be easilywashed away when applying in digestion, its permeability and themechanical strength were vital for filling the GA activated hybridsilica particles. Huang et al.’s research [18] showed that the ace-tonitrile/dodecanol (v/v) ratio could affect the specific surface area,average pore diameter and specific pore volume of p-MAA-EGDMAmonolith, which may be beneficial in applications. So the acetoni-trile/dodecanol (v/v) ratio from 1:3 to 1:5 was investigated, and theobtained results showed that the monolith structure had a highmechanical strength when the acetonitrile/dodecanol (v/v) ratiowas 1:3, but the permeability was so poor that a high pressurewas needed to push the liquid through the layer. When acetoni-trile/dodecanol (v/v) ratio was 1:5, the permeability of the monolithwas improved, but the rigidity of monoliths decreases so that thelayer was easily destroyed. The good p-MAA-EGDMA layer with sat-isfied rigidity and low-back pressure of layer was obtained whenthe ratio of acetonitrile/dodecanol (v/v) was 1:4. So, 1:4 of acetoni-trile/dodecanol was chosen as optimal condition for synthesis ofp-MAA-EGDMA, and the SEM image of p-MAA-EGDMA monolithpresented in Fig. 3S demonstrated that the monolith was of homo-geneous and porous structure with micro pores, which was helpfulto magnify the immobilized trypsin reactors for digesting proteinsin large scale.

3.2. The application of large-volume IMER in BSA digestion

To produce more specific peptides, the concentration of GA andthe given amount of trypsin for preparation of large-volume IMERwere investigated and the obtained results were shown in Fig.4S. The sequence coverage decreased with the increasing of theconcentration of GA as well as the given amount of trypsin (SeeTables 3S–16S supplementary materials). The reasons might be thesteric hindrance effect of trypsin on silica particles with the givenamount of trypsin increasing and the bis-aldehyde crosslinker ren-ders crosslinking among particles inevitable [26], which wouldthen lead to the loss of the aldehyde groups to be used for thesuccessive coupling reactions with the amino group of trypsinmolecules.

For most studies, the digestion performance was evaluated byinvestigating the sequence coverage. In our test, the sequence cov-erage presents in a low level (the highest sequence coverage was20%, Fig. 4Sa) because of the simple digestion condition (only inan incubator for 10 min) and the undigested protein obviouslyexists (in retention time of 33–36 min) (Reviewer 2). But theimproved sequence coverage does not necessarily mean betterdigestion efficiency unless quantitative analysis is carried out [27].The abundance of peptides is determined as an alternative way toquantitatively analyze peptides. The main peptides peaks emergefrom 9 to 32 min on LC–MS spectrum in the optimized separa-tion condition. As shown in Fig. 4Sb–d, the profiles of sequencewere the same at 101–105, 156–160, 205–209, 212–218, 229–232,257–263 and 473–451 of the amino position of BSA and the abun-dance of peptides decreased with the GA concentration increasing.

However, the results of further experiments indicated that thesequence number of peptides produced from large-volume IMERprepared in the conditions of 10 mg/mL (Fig. 4Sc) and 15 mg/mL(Fig. 4Sd) of given amount of trypsin was nearly stable with the GA

16 G. Ruan et al. / J. Chromatogr. B 967 (2014) 13–20

Table 1The common peptides obtained from digestion of BSA in different IMERs prepared conditions.

Sequence number Amino acid sequence Peptides abundance in different IMERsprepared conditionsa

The ratio of maximum andminimum abundanceb

Maximum Minimum

101-105 VASLR 3b-3 3b-4 68.3156-160 KFWGK 3b-5 3c-4 95.3205-209 IETMR 3b-2 3d-3 461.3212-218 VLTSSAR 3c-3 3b-3 326.4229-232 FGER 3d-3 3d-4 207.3257-263 LVTDLTK 3b-4 3b-3 2925.2473-451 KVPQVSTPTLVEVSR 3d-1 3c-3 353.9

f peptides/the minimum abundance of peptides.

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Amino acid sequence Sequence number Frequency

IETMR 205-209 10VLTSSAR 212-218 10FGER 229-232 10LSQKFPK 242-248 10LVTDLTK 257-263 10ECCDKPLLEK 300-309 10KVPQVSTPTLVEVSR 437-451 10SLGK 452-455 10CASIQK 223-228 9LKECCDKPLLEK 298-309 9SHCIAEVEK 310-318 9LCVLHEKTPVSEKVTK 483-498 9AEFVEVTK 249-256 8HLVDEPQNLIK 402-412 8TPVSEKVTK 490-498 7LVVSTQTALA 600-609 7KFWGK 156-160 6

a Conditions for preparing IMERs were as the conditions of Fig. 4S.b The ratio of maximum and minimum abundance = the maximum abundance o

oncentration increasing, because the excessive enzyme loading onarge-volume IMER blocked the interaction between enzyme androtein [28].

To investigate the selectivity of large IMER, common peptidesrom large-volume IMER prepared in different conditions wereicked out and the ratio of maximum and minimum abundancef peptides with amino acid sequence was calculated. The resultsTable 1) showed that the fragment of “LVTDLTK” exhibited the

aximum ratio of 2925.2, illustrating the potential high selectiv-ty of large-volume IMER. In addition, the abundance of differenteptides by using the large-volume IMERs reached the maximumnder different experimental conditions, e.g. the abundance of pep-ide “LVTDLTK” was max in the condition of 5 mg/mL of trypsin and.4% of GA (Fig. 4Sb-4) but minimum in the condition of 5 mg/mLf trypsin and 0.2% of GA (Fig. 4Sb-3). It suggests the possibility toelectively produce peptides with specific sequence via controllinghe preparation of large-volume IMER.

.3. Reusability of large-volume IMER

The reusability of the large-volume IMER is important forbtaining more peptides with specific sequence desired. Ten runs ofSA digestion with the large-volume IMER were investigated, andig. 1 illustrates the peptides profile of the 1st (Fig. 1 A) run and the

0th (Fig. 1 B) run (see Table 18S-27S in supplementary materials).he stability of large-volume IMER is satisfactory after ten runs. Theommon peptides in ten runs of BSA digestion with large-volume

ig. 1. The comparison of LC-ESI-MS between the first and tenth run of BSA digestionith large-volume of IMER. (A) The first run, (B) the tenth run. Digestion condition:

.1% (w/v) GA, 10 mg/mL of trypsin, 1 mL 1 mg/mL BSA, 10 min, 50 ◦C.

AFDEK 524-528 6GACLLPK 198-204 5

IMER were list in Table 2, and 8 of the common peptides couldbe observed. This could further demonstrate the stability of thelarge-volume IMER, which would be qualified in specific sequencepeptides production.

3.4. Regeneration of IMERs

In general, when using GA as crosslinker covalently immobi-lized of trypsin to prepared IMERs, a suitable reducing agent likeNaBH3CN was added to reduce Schiff base double bonds –C N– to–C-N–, thus enhancing the stability of immobilized trypsin [29]. Inour experiment, –C N– was broken by adding HCl solution becausethe reverse reaction of Schiff base was easily carried out in the acidcondition, resulting to R–CHO and R–NH2. The residual –NH2 onsilica surface could be activated by adding GA and then the IMERwas regenerated via fresh trypsin flowed through. As Fig. 2 shown,the aldehyde carbonyl group on the GA modified silica particles isclearly evident as the peak at 1640 cm−1 (Fig. 2A). After immobiliz-ing trypsin on modified silica particles (Fig. 2B), many split peaksemerge in the range of 900–1500 cm−1. However, these peaks dis-appear or become weaken (Fig. 2C) when we treated the IMER withHCl, which means that the enzyme and GA coated on silica particleswere successfully removed in the condition of HCl acid. Fig. 2B andD illustrates that the IMER could be well recovered.

As shown in Fig. 3, after the BSA was digested with HCl treated

IMER (Fig. 3B), some peaks (12–23 min) disappeared while theintensity of the remained ones (23–30 min) were significantlyreduced in contrast to the first use of IMER (Fig. 3A). This illustratedthis method to regenerate IMER was feasible. On the other hand,

G. Ruan et al. / J. Chromatogr. B 967 (2014) 13–20 17

F ) 0.1%

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ig. 2. The IR spectrum of relative products in the process of regenerating IMER. (A mL of trypsin (5 mg/mL). (C) The IMER treated with 5 mL of HCl (0.01 M). (D) Reg5 mg/mL).

n contrast to the first use of IMER (Fig. 3A), the regenerated IMERFig. 3C) obtained similar peptide profiles, demonstrating that theegenerated IMER had same cleavage sites, which was meaningful

or bioactive peptides production. Details information is shown inable 3. Although different peptide sequences were yielded due tohe release of new cleavage sites of trypsin after regeneration, 14

able 3dentification results of BSA digested by IMER* and regenerated IMER.

Number of peptides Amino acids

The first use of IMER 16 104

The regenerated IMER 22 154

* Conditions for IMER preparation: hybrid silica particles were reactivated by 0.1% Gmmobilized on.

GA modified amino activated silica particles. (B) IMER prepared in the condition ofed IMER undergo being reactivated by 2 mL of 0.1% GA and the 2 mL fresh trypsin

common peptides (accounting for 87.5% of first use) were producedboth in IMER and regenerated IMER. Moreover, the improvingnumber of peptides, amino acids, specific peptides and sequence

coverage (from 17% to 25%) were obtained with regenerated IMER.It is likely that HCl also broken the previous cross linking GAon silica surface, and more sites were released for the following

Unique peptides Common peptides Sequence coverage (%)

10 14 1717 14 25

A and the 2 mL fresh trypsin (5 mg/mL, 100 mM phosphate buffer, pH 8.0) was

18 G. Ruan et al. / J. Chromatogr. B 967 (2014) 13–20

Fig. 3. The evaluation of regeneration property of large-volume IMER. (A) The first use, (B) HCl treated, (C) after regenerating. Digestion condition: 1 mL 1 mg/mL BSA, 10 min,50 ◦C.

Fig. 4. The LC–MS of non-digested and digested Cyt-c. Digestion condition: 1 mL 1 mg/mL Cyt-c, 10 min, 50 ◦C. The more information of T1–T4 peak was given in Table 28S.

G. Ruan et al. / J. Chromatogr. B 967 (2014) 13–20 19

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ig. 5. The LC–MS of ginkgo solution filtered through the IMER column (A), digeondition: 1 mL ginkgo extraction solution, 10 min, 50 ◦C. The ginkgo hydrolysateeptides identification was given in Table 29S-30S.

mmobilization. These results indicated this regeneration methodot only kept the stability of peptide production but also improvedhe efficiency of IMER.

.5. Application of large-volume IMER in Cyt-c and ginkgoigestion

To investigate the digestion performance of large-volume IMERor other protein, Cyt-c and ginkgo was chosen as model sub-trate for the IMER digestion. Cyt-c on-column digestion resultsre shown in Fig. 4. Although the sequence coverage was low (19%,een in Table 28S), compared with intensity of peaks of undigestedyt-c (Fig. 4A), the significant intensity of peaks of the digestedyt-c (Fig. 4B) could still demonstrate that the large-volume IMERas successfully applied in fast, efficient and potential orientedigestion. The database searching illustrated the species of protein

n ginkgo were ginnacin and legumin (Tables 29S and 30S). Fig. 5resented the on-column digestion results of ginkgo. Although theequence coverage of the ginkgo solution that filtered through theMER column during the process of digestion reached both 51%or ginnacin and legumin (Table 29S), the peptides peaks on theC-MS (Fig. 5A) were not significant. When 1 mL of 20% (v/v) ACNqueous solution was applied to elute the ginkgo hydrolysates, sig-ificant peptides peaks emerges at 10–17 min of retention timen LC-MS (Fig. 5B). Moreover, the sequence coverage of two mainrotein of ginkgo, ginnacin and legumin, was 56% and 55%, respec-ively (Table 30S). On the other hand, the less peaks of LC-MS ofon-digested ginkgo (Fig. 5C) and the results of non-identified pep-ides (sequence coverage was 0%) proved most of peptides weredsorbed on the IMER column. This illustrated the potential utilityf the large-volume IMER in isolating real life bioactive peptides

Reviewer 2).

Although the feasible application and superiority of the large-olume IMER were proved, the separation and the identification ofioactivity peptides from practical digestion was still a hard task.

inkgo elution from the IMER column (B) and non-digested ginkgo (C). Digestione eluted with 1 mL of 20% (v/v) ACN aqueous solution. The more information of

Our further studies will focus on the precise analysis of bioactivepeptides.

4. Conclusion

In this study, we develop a novel large-volume IMER for diges-tion of proteins Result show that the large-volume IMERs preparedin the condition of 10 and 15 mg/mL trypsin could obtain peptideswith stable specific sequence. Ten runs of BSA digestion on IMERindicated that the sequence did not have any obvious changes.In addition, an approach to regenerate IMER was proposed anddemonstrated by using HCl to break –C N– and rebuilding by thefollowing GA activated, and the efficiency of IMER was improvedafter regeneration. Fourteen common peptides (accounting for87.5% of first use) were produced both with first use and regen-erated IMER. Finally, this IMER was proved to successfully apply infast and efficient digestion of real samples-ginkgo. The sequencecoverage of two main protein of ginkgo, ginnacin and legumin, was56% and 55%, respectively. Although the sequence coverage of BSAand Cty-c were low when comparing the conditional IMER [30],the method of regenerating was proposed. Base on this method,our work continued to design another new regenerating IMER andnow some interesting results were obtained, including higher cov-erage and better stability and durability (Reviewer 2). The methodof regenerating IMER is expected to open up a new possibility forthe preparation of bioactive peptides as well as the regeneration ofimmobilized enzyme.

Acknowledgements

The authors would like to thank the National Natural Sci-

ence Foundation of China for financial supporting under ContractNo. 21065003 and No. 21265004. Also, thanks the Natural Sci-ence Foundation from Guangxi Zhuang Autonomous Region (No.2012GXNSFAA053030) and the scientific research project from

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ducation department of Guangxi Zhuang Autonomous Region (No.00911MS103), respectively.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.jchromb.014.07.008.

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