Supplematary materials of TJ12P2 revised

Supplemental information

Development of Glypican-3-Specific Binding Peptide using in vivo and in vitro

Two-step Phage Display Screening for PET Imaging of Hepatocellular Carcinoma

Yushuang Qin#, 1, Siyuan Cheng#, 1, Yesen Li2, Sijuan Zou1, Minglong Chen3, Dongling

Zhu1, Shi Gao3, Hua Wu2, Lei Zhu*,4, Xiaohua Zhu*,1

1. Department of Nuclear Medicine and PET, Tongji Hospital, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan 430000, China;

2. Department of Nuclear Medicine, The First Affiliated Hospital of Xiamen

University, Xiamen 361003, China;

3. Department of Nuclear Medicine, China-Japan Union Hospital, Jilin University,

Changchun 130033, China;

4. Departments of Surgery, Emory University School of Medicine, Atlanta, Georgia

30322, United States.

Corresponding Author

* To whom correspondence should be addressed. E-mail: [email protected] (X.Z.)

and [email protected] (L.Z.)

# Yushuang Qin and Siyuan Cheng contributed equally to the article.

Electronic Supplementary Material (ESI) for Biomaterials Science.This journal is © The Royal Society of Chemistry 2020

Figure S1. Synthesis of TJ12P2. (a). Chemical structure of TJ12P2 with the sequence

of SNDRPPNILQKR. (b). Purity of TJ12P2 by analytical HPLC. The retention time of

TJ12P2 is 18.51 min. (c). Mass spectrum analysis of molecular weight of TJ12P2,

which is 1425.76 Da.

a

6 8 10 12 14 16 18 20 22

0

400

800

1200

1600

2000

Val

ue

(mA

U)

Time (min)

GPC3-TJ12P-2

QYS-2_150714153055 #145-174 RT: 0.66-0.79 AV: 30 NL: 1.87E8T: FTMS + p ESI Full ms [100.00-1000.00]

440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Abu

ndan

ce

479.5944z=3

718.8870z=2

492.2428z=3

473.5911z=3

580.8868z=3

530.2409z=3

503.7917z=?

729.8780z=2

654.8580z=2

560.8373z=2

709.8821z=2

450.5834z=3

669.5306z=3

618.3499z=2

640.8366z=2

698.4284z=1

592.3720z=3

753.1826z=?

TJ12P2

b

c

Figure S2. Synthesis of Biotin-TJ12P2. (a). Chemical conjugation of Biotin onto the

N-terminal of TJ12P2. (b). Mass spectrum analysis of molecular weight of TJ12P2,

which is 1662.8 Da. (c). Purity of Biotin-TJ12P2 by analytical HPLC. The retention

time of Biotin-TJ12P2 is 22 min.

0 5 10 15 20 25 30 35 40

0

200

400

600

Val

ue(m

AU

)

Time (min)

Biotin-TJ12P2

1-PEP-QYS #62-76 RT: 0.67-0.79 AV: 7 NL: 2.02E6T: FTMS + p ESI Full ms [200.00-3000.00]

780 790 800 810 820 830 840 850 860 870 880 890 900 910m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bunda

nce

832.4204z=2

833.9226z=2

843.4109z=2

851.3945z=2

823.9165z=2

836.1599z=1

796.0019z=3

908.8915z=2

782.4027z=?

895.9592z=?

801.0107z=?

890.8021z=3

815.8375z=2

859.1212z=?

864.3684z=1

875.9377z=?

a

b c

Figure S3. Synthesis of Cy5.5-TJ12P2. (a). Chemical conjugation of Cy5.5-NHS onto

the N-terminal of TJ12P2. (b). Mass spectrum analysis of molecular weight of TJ12P2,

which is 1662.8 Da. (c). Purity of Cy5.5-TJ12P2 by analytical HPLC. The retention

time of Biotin-TJ12P2 is 33.12 min.

0 5 10 15 20 25 30 35 40

0

200

400

600

800

Va

lue

(m

AU

)

Time (min)

CY5.5-GPC3-TJ12P-2.

CY5-PEP-QYS #64-75 RT: 0.69-0.80 AV: 6 NL: 4.10E5T: FTMS + p ESI Full ms [200.00-3000.00]

600 650 700 750 800 850 900 950 1000 1050m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bun

dan

ce

668.0343z=3

610.1847z=1

576.8459z=? 654.3323

z=1 832.4205z=2

712.8206z=?

684.2032z=1

618.9076z=? 1001.5469

z=2592.8199

z=?738.8300

z=1978.7537

z=?880.1331

z=3807.4714

z=3780.4787z=3

1058.0434z=2

900.8127z=?

931.1152z=?

a

b c

Cy5.5-TJ12P2

Figure S4. Synthesis of NOTA-TJ12P2. (a). Chemical structure of NOTA-TJ12P2.

(b). Purity of NOTA-TJ12P2 by analytical HPLC. The retention time of Biotin-TJ12P2

is 20.20 min. (c). Mass spectrum analysis of molecular weight of TJ12P2, which is 1722

Da.

Figure S5. Synthesis of scramble TJ12P2 peptide. (a). Chemical structure of

scramble-TJ12P2. (b). Purity of Cy5.5-TJ12P2 by analytical HPLC. The retention time

of scramble-TJ12P2 is 18.40 min. (c). Mass spectrum analysis of molecular weight of

scramble-TJ12P2, which is 1436.76 Da.

Figure S6. Synthesis of Biotin-scrambled-TJ12P2. (a). Chemical conjugation of

Biotin onto the N-terminal of scrambled-TJ12P2. (b). Mass spectrum analysis of

molecular weight of scrambled-TJ12P2, which is 1662.8 Da. (c). Purity of Biotin-

scrambled-TJ12P2 by analytical HPLC. The retention time of Biotin- scrambled-

TJ12P2 is 21.3 min.

Figure S7. Synthesis of Cy5.5-scrambled-TJ12P2. (a). Chemical conjugation of

Cy5.5-NHS onto the N-terminal of scrambled-TJ12P2. (b). Mass spectrum analysis of

molecular weight of Cy5.5-scrambled-TJ12P2, which is 2002.09 Da. (c). Purity of

Cy5.5-scrambled-TJ12P2 by analytical HPLC. The retention time of Biotin-TJ12P2 is

32.97 min.

0 5 10 15 20 25 30 35 40

0

200

400

Val

ue

(mA

u)

Time (min)

Cy5.5-SCR-GPC3-TJ12P-2

CY5-PEPTIDE #131-156 RT: 0.59-0.70 AV: 26 NL: 4.24E6T: FTMS + p ESI Full ms [200.00-3000.00]

600 650 700 750 800 850 900 950 1000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bund

ance

659.2865z=1

610.1836z=1

668.3614z=3

624.8464z=1 654.3313

z=1719.3806

z=2682.8049

z=?637.3046

z=1 702.8625z=?

760.8212z=?

803.5420z=1740.7637

z=? 878.7349z=?

838.8365z=?

896.7959z=?

820.7768z=1

954.7545z=1

981.2781z=1936.6939

z=?

798.7222z=1 1014.7104

z=1

a

cb

Figure S8. HPLC analysis of the stability of 18F-AlF-NOTA-TJ12P2 in physiological

buffer at 37°C for 4 hours.

0 10 20 30 40

200

400

600

800

Retention Time (min)

Co

un

t

Figure S9. Micro-PET/CT imaging of 18F-AlF-NOTA-TJ12P1 targetability in

HepG2 tumor bearing mouse model. White arrow points to the HepG2 tumor and red

circle indicates the normal liver.