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Supporting Information

One-Pot Synthesis of Trypsin-Based Magnetic Metal-Organic

Frameworks for Highly Efficient Proteolysis

Chao Zhong,a♀ Zhixian Lei,a♀Huan Huang,a Mingyue Zhang,a Zongwei Cai,b and

Zian Lina,*

a. Ministry of Education Key Laboratory of Analytical Science for Food Safety and

Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for

Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116,

China

b. State Key Laboratory of Environmental and Biological Analysis, Department of

Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong

Kong, SAR, P. R. China

Corresponding author: Zian Lin;

Postal address: College of Chemistry, Fuzhou University,

Fuzhou, Fujian, 350116, China

Fax: 86-591-22866135

E-mail: zianlin@fzu.edu.cn (Z.A. Lin);

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2020

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Experimental section

Chemicals and reagents. Zinc nitrate hexahydrate (Zn(NO3)2), ferric chloride

hexahydrate (FeCl3·6H2O), sodium acetate anhydrous (NaOAc), sodium citrate

dihydrate (Na3Cit·2H2O), ethylene glycol (EG) and ethanol were purchased from

Sinopharm Chemical Reagent, Co., Ltd. (Shanghai, China). 2-methylimidazole

(HmIm), trifluoroacetic acid (TFA), TPCK-treated trypsin from bovine pancreas and

N-α-benzoyl-L-arginine ethyl ester (BAEE) were obtained from Aladdin Chemistry

Co., Ltd (Shanghai, China). Cytochrome C (Cyt-C) from horse heart, bovine serum

albumin (BSA) and horse radish peroxidase (HRP) were purchased from Shanghai

Lanji Co., Ltd. (Shanghai, China). Iodoacetic acid (IAA) and 1,4-dithiothreitol (DTT)

were purchased from Sigma-Aldrich Chemical Co., Ltd. (Shanghai, China). Healthy

human serum was kindly donated by Fujian Provincial Governmental Hospital

(Fuzhou, China). The deionized water used in all experiments was treated with a

Milli-Q purification system (Millipore, USA). All other reagents were of analytical

grade or better.

Preparation of CA-Iron Oxide Magnetic Nanoparticles. The synthesis of CA-

iron oxide magnetic nanoparticles was described elsewhere.1 In brief, FeCl3·6H2O

(1.35 g, 8.0 mmol) and Na3Cit·2H2O (0.22 g, 0.68 mmol) were first dissolved in EG

(20 mL), afterward, NaOAc (1.20 g) was added with stirring. The mixture was stirred

vigorously for 30 min and then sealed in a Teflon-lined stainless-steel autoclave (50

mL capacity). The autoclave was heated at 200 °C and maintained for 10 h, and then

allowed to cool to room temperature. After that, the black products were washed with

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ethanol and deionized water for several times. Finally, the prepared Fe3O4 was dried.

Preparation of Iron oxide@ZIF-8. In brief, HmIm (4.1g) was dissolved in

deionized water (40 mL) and adjusted to pH 9.4 by HCl (12 M, 0.1 mL). Then the

solution was mixed with a separate aqueous solution (2 mL) containing iron oxide (50

mg), afterward, zinc nitrate solution (310 mM, 4 mL) was added with stirring. After

continuous stirring for 1 h at room temperature, the products were collected with a

magnet and washed with ammonium bicarbonate (NH4HCO4) buffer solution (50 mM,

pH 8.0), and freeze-dried for further characterizations.

Characterization. In order to observe the morphologies and detailed structures

of the CA- iron oxide nanoparticle and CA-iron oxide @ZIF-8@Trypsin, scanning

electron micrographs (SEM) and transmission electron micrographs (TEM) was

performed on a Hitachi SU8020 scanning electron microscope and FEI Tecnai G20

transmission electron microscope, respectively. Fourier-transform infrared (FT-IR)

spectra were recorded from samples in KBr pellets using a Thermo Nicolet 6700 FT-

IR instrument within the range of 400-4000 cm-1. The crystal structures of the

products were determined by a Shimadzu D/Max-2500 powder X-ray diffraction

(PXRD) analysis diffractometer. Surface area and pore size analysis of the products

were performed by Brunauer-Emmett-Teller (BET) and Barrett-JoynerHalenda (BJH)

methods using Micromeritics ASAP2020 physisorption analyzer. Thermogravimetric

analysis (TGA) was carried out on a TAQ600 thermogravimetric analyzer in the range

of 30-600 °C under an air atmosphere (heating rate of 5 °C/min). Magnetic

characterization was performed on a quantum design magnetic properties

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measurement system (MPMS) XL-7 superconducting quantum interference device

(SQUID) magnetometry at room temperature. The activity of the trypsin in the

products were measured by using a Shimadzu UV2550 UV-vis spectrophotometer.

Proteolytic peptide mixtures were analyzed by Bruker autoflex matrix-assisted laser

desorption/ionization time of flight mass spectrometer (MALDI-TOF MS).

Pre-treatment of Protein Samples. Prior to digestion, HRP was denatured in a

100 ºC water bath for 10 min, BSA and the healthy human serum were reduced and

alkylated using following protocol: 1.0 mg BSA in 1.0 mL of 50 mM NH4HCO3

buffer solution (pH 8.0) (or 10 μL human serum in 590 μL of 50 mM NH4HCO3

buffer solution (pH 8.0), which corresponds to 600 μg total proteins by Brandford

protein assay (Bio-Rad, Catalog. No. P0006) using BSA a standard was denatured in a

100 ºC water bath for 10 min and then reduced in 10 mM DTT for 1h at 56 ºC. When

cooled to room temperature, cysteines of proteins were alkylated in the dark in 20

mM IAA for 40 min at 37 ºC.

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Fig. S1 SEM images of (A) iron oxide and (B) iron oxide@ZIF-8@Trypsin.

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Fig. S2 EDS spectra of (A) iron oxide and (B) iron oxide@ZIF-8@Trypsin.

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Fig. S3 XRD patterns of iron oxide (experiment, blue), iron oxide@ZIF-8@Trypsin (experiment, black), ZIF-8 (experiment, green) and ZIF-8 (simulation, red).

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Fig. S4 the TEM of iron oxide@ZIF-8@Trypsin (A and B) and iron oxide@ZIF-8 (C and D) before and after thermal treatment.

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Fig. S5 the nitrogen sorption isotherms for iron oxide@ZIF-8@Trypsin composite, ZIF-8@Trypsin and ZIF-8 before and after thermal treatment.

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Fig. S6 the pore-size distributions of iron oxide@ZIF-8@Trypsin composite, ZIF-8@Trypsin and ZIF-8 before and after thermal treatment, calculated by the DFT method.

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Fig. S7 pore-size distributions of iron oxide@ZIF-8@Trypsin, ZIF-8@Trypsin and ZIF-8 before and after thermal treatment, calculated by the DFT method.

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Fig. S8 the catalytic kinetics and reaction rate (inset) of the ester hydrolysis of BAEE at pH 8.0 by the (A) Iron oxide@ZIF-8@Trypsin (synthesized at pH 9.4), (B) free trypsin, (C) iron oxide@ZIF-8@Trypsin (synthesized at pH 11.6) and (D) Iron Oxide@ZIF-8.

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Table. S1 Comparison of the proteolytic efficiency of the immobilized micro-enzyme reactor and free trypsin for Cyt-c digestion.

Group Position Mass (Da) Sequence Freetrypsin

Immobilized trypsin

Enzymolysis time 12 h 5 minMatched peptides 17 17 Sequences coverage of amino acid 89.4% 91.3%

1 88-91 533.3027 (K)KTER(E) √ ×2 23-27 543.0490 (K)GGKHK(T) √ ×3 100-104 562.9186 (K)KATNE(-) × √4 1-5 590.7986 (-)GDVEK(G) √ √5 56-60 604.6974 (K)GITWK(E) √ √6 9-13 634.3489 (K)IFVQK(C) √ ×7 74-79 677.0404 (K)YIPGTK(M) √ √8 80-86 779.1224 (K)MIFAGIK(K) √ √9 73-79 806.1212 (K)KYIPGTK(M) × √10 54-60 862.1017 (K)NKGITWK(E)+Oxidation × √11 80-87 907.2043 (K)MIFAGIKK(T) × √12 14-22 1020.1396 (K)CAQCHTVEK(G) × √13 92-100 1109.1315 (R)EDLIAYLKK(A)+Oxidation × √14 28-38 1168.1861 (K)TGPNLHGLFGR(K) √ √15 28-39 1296.7716 (K)TGPNLHGLFGRK(T) √ ×16 89-99 1350.2082 (K)TEREDLIAYLK(K) √ √17 40-53 1470.1142 (K)TGQAPGFTYTDANK(N) √ √18 61-72 1495.5744 (K)EETLMEYLENPK(K) √ ×19 39-53 1598.1304 (R)KTGQAPGFTYDANK(N) √ √20 61-73 1623.1348 (K)EETLMEYLENPKK(Y) √ √21 9-22 1632.9519 (K)IFVQKCAQCHTVEK(G) √ √22 40-55 1713.8866 (K)TGQAPGFTYTDANKNK(G) √ ×23 56-72 2079.9579 (K)GITWKEETLMEYLENPK(K) √ √

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Table. S2 Comparison of the proteolytic efficiency of the immobilized micro-enzyme reactor and free trypsin for HRP digestion.

Group Position Mass (Da) Sequence Freetrypsin

Immobilized trypsin

Enzymolysis time 12 h 5 minMatched peptides 16 21 Sequences coverage of amino acid 56.8% 76.6%

1 179-183 681.0668 (R)FIMDR(L) × √2 32-38 743.2069 (R)IAASILR(L) √ √3 76-82 803.1765 (R)GFPVIDR(M) √ √4 85-93 903.1676 (K)AAVESACPR(T) √ √5 225-232 935.2201 (R)TPTIFDNK(Y) √ √6 20-27 959.2377 (R)DTIVNELR(S) √ √7 66-75 1022.1429 (K)DAFGNANSAR(G) √ √8 233-241 1185.2392 (K)YYVNLEEQK(G) √ √9 150-159 1199.2044 (K)DSFRNVGLNR(S)+Oxidation √ √10 63-75 1380.2003 (R)TEKDAFGNANSAR(G) × √11 160-174 1475.2414 (R)SSDLVALSGGHTFGK(N) × √12 284-298 1586.2746 (R)MGNITPLTGTQGQIR(L) √ √13 207-224 1916.9029 (R)GLCPLNGNLSALVDFDLR(T) × √14 284-302 2072.1533 (R)MGNITPLTGTQGQIRLNCR(V) √ √15 207-232 2848.7813 (R)GLCPLNGNLSALVDFDLRTPT

IFDNK(Y)× √

16 1-19 3321.8386 (-)QLTPTFYDN(*)SCPNVSNIVR(D)

√ ×

17 265-283 3386.8029 (R)SFAN(*)STQTFFNAFVEAMDR(M)

× √

18 233-264 3668.8947 (K)YYVNLEEQKGLIQSDQELFSSPNATDTIPLVR(S)

× √

19 242-274 3671.8947 (K)GLIQSDQELFSSPN(*)ATDTIPLVR(S)

√ √

20 39-62 3894.5267 (R)LHFHDCFVNGCDASILLDN(*)TTSFR(T)

√ √

21 1-19(85-93)

4221.6300 (-)QLTPTFYDNSC[AAVESACPR]PN(*)VSNIVR(D)

√ √

22 184-206 4986.5521 (R)LYN(*)FSNTGLPDPTLN(*)TTYLQTLR(G)

√ √

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Table. S3 Comparison of the proteolytic efficiency of the immobilized micro-enzyme reactor and free trypsin for BSA digestion.

Group Position Mass (Da) Sequence Freetrypsin

Immobilized trypsin

Enzymolysis time 12 h 5 minMatched peptides 35 40 Sequences coverage of amino acid 51% 51.7%

1 456-459 432.2173 (K)VGTR(C) √ √2 434-436 439.2062 (R)YTR(K) √ √3 20-23 479.7142 (R)GVFR(R) × √4 229-232 508.1818 (K)FGER(A) √ √5 25-28 515.3452 (R)DTHK(S)+Oxidation √ ×6 157-160 537.2596 (K)FWGK(Y) √ ×7 101-105 545.2538 (K)VASLR(E) √ √8 219-222 572.4198 (R)QRLR(C) √ ×9 524-528 609.2671 (K)AFDEK(L) √ ×10 205-209 649.1862 (K)IETMR(E) √ √11 118-122 659.2516 (K)QEPER(N) × √12 156-160 665.2568 (K)KFWGK(Y) √ √13 236-241 689.2402 (K)AWSVAR(L) √ √14 29-34 712.2293 (K)SEIAHR(F) × √15 341-346 752.4530 (K)NYQEAK(D) √ ×16 452-459 817.2632 (R)SLGKVGTR(C) × √17 229-235 821.1337 (K)FGERALK(A) √ ×18 242-248 847.4935 (R)LSQKFPK(A) √ ×19 483-489 899.2312 (R)LCVLHEK(T)

+Carboxymethyl (C)× √

20 37-44 990.6009 (K)DLGEEHFK(G)+Oxidation × √21 205-211 906.0414 (K)IETMREK(V) × √22 161-167 927.2942 (K)YLYEIAR(R) √ √23 598-607 1002.7330 (K)LVVSTQTALA(-) × √24 233-241 1017.3519 (R)ALKAWSVAR(L)+Oxidation √ √25 123-130 1058.7168 (R)NECFLSHK(D)

+Carboxymethyl(C)+Na+

× √

26 310-318 1073.1940 (K)SHCIAEVEK(D)+Carboxymethyl(C)

× √

27 499-507 1140.5330 (K)CCTESLVNR(R)+Carboxymethyl(C)

√ ×

28 257-266 1153.4104 (K)LVTDLTKVHK(E) × √

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Group Position Mass (Da) Sequence Freetrypsin

Immobilized trypsin

29 66-75 1163.6033 (K)LVNELTEFAK(T) √ √30 460-468 1168.3798 (R)CCTKPESER(M)

+Carboxymethyl(C)× √

31 257-266 1170.5031 (K)LVTDLTKVHK(E)+Oxidation √ ×32 223-232 1233.2722 (R)CASIQKFGER(A)

+Carboxymethyl(C)× √

33 35-44 1249.4506 (R)FKDLGEEHFK(G) √ ×34 337-346 1255.6131 (K)DVCKNYQEAK(D)

+Carboxymethyl (C)× √

35 402-412 1305.3748 (K)HLVDEPQNLIK(Q) √ √36 286-297 1386.8203 (K)YICDNQDTISSK(L) × √37 569-580 1399.5090 (K)TVMENFVAFVDK(C) √ ×38 198-209 1406.4416 (K)GACLLPKIETMR(E)

+Oxidation+Carboxymethyl (C)√ ×

39 89-100 1420.4909 (K)SLHTLFGDELCK(V) +Carboxymethyl (C)

√ √

40 421-433 1479.4739 (K)LGEYGFQNALIVR(Y) √ √41 347-359 1567.4495 (K)DAFLGSFLYEYSR(R) × √42 437-451 1639.5688 (R)KVPQVSTPTLVEVSR(S) √ √43 249-263 1691.6578 (K)AEFVEVTKLVTDLTK(V) × √44 267-280 1776.2269 (K)ECCHGDLLECADDR(A)

+Carboxymethyl(C)+Na+

× √

45 508-523 1881.5085 (R)RPCFSALTPDETYVPK(A) +Carboxymethyl(C)

√ √

46 529-544 1908.6099 (K)TVMENFVAFVDK(C) √ ×47 139-155 2020.9815 (K)LKPDPNTLCDEFKADEK(K)

+Carboxymethyl (C) √ ×

48 588-607 2092.6876 (K)EACFAVEGPKLVVSTQTALA(-)+Carboxymethyl(C)

× √

49 264-280 2149.5424 (K)VHKECCHGDLLECADDR(A)+Carboxymethyl(C)+Oxidation

× √

50 300-318 2364.0826 (K)ECCDKPLLEKSHCIAEVEK(D)+Oxidation+Carboxymethyl (C)

√ ×

51 267-285 2272.6523 (H)ECCHGDLLECADDRADLAK(Y)+Carboxymethyl(C)+Na+

× √

52 402-420 2379.2326 (K)HLVDEPQNLIKQNCDQFEK(L) +Carboxymethyl (C)+Na+

× √

53 508-528 2430.4550 (R)RPCFSALTDETYVPKAFDEK(L)+Oxidation

× √

54 319-340 2458.7702 (K)DAIPENLPPLTADFAEDKDVCK(N)

√ ×

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Group Position Mass (Da) Sequence Freetrypsin

Immobilized trypsin

55 184-204 2489.6306 (K)YNGVFQECCQAEDKGACLLPK(I)

√ ×

56 45-65 2493.5458 (K)GLVLIAFSQYLQQCPFDEHVK(L)

√ ×

57 66-88 2609.9251 (K)LVNELTEFAKTCVADESHAGCEK(S)

× √

58 499-523 3003.5743 (K)CCTESLVNRRPCFSALTPDETYVPK(A)+Carboxymethyl (C)

√ √

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