1

연구 결과 보고서
(Tissue 시료의 프로테오믹스 분석)

주문자 정보 : 인제대학교 한진

분석 시행 기관 : ㈜ 이바이오젠

보고서 발행 일자: 2023년 10월 20일

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1. 연구제목

Tissue 시료의 프로테오믹스 분석

2. 연구목표

Tissue 시료의 프로테오믹스 분석을 위해, UPLC / Q-Exactive 장비를 이용하여 15 개의 시료를 총 15 번

run의 분석을 진행함.

3. 연구방법

3.1. FASP digestion

1) BCA assay를 BCA assay를 통해 측정한 단백질 정량값을 바탕으로 FASP digestion을 진행함. 초기량은
100 μg으로 시작함.

2) 500 mM TCEP(Final conc. 5 mM TCEP)를 첨가한 후, 37 ℃에서 30 분 동안 incubation 하여 단백질을

reduction 시키고, 15 분 동안 14,000 g로 내림.

3) 500 mM IAA(Final conc. 50 mM)을 첨가한 후, 30 분 동안 25 ℃의 어두운 곳에서 incubation 하여 단백

질을 alkylation 시키고, 15 분 동안 14,000 g로 내림.

4) 8 M Urea(in 0.1 M Tris/HCl, pH 8.5) 200 μL를 첨가한 후, 15 분 동안 14,000 g로 내림. (3회 반복)

5) 50 mM ABC 100 μL를 첨가한 후, 15 분 동안 14,000 g로 내림. (3회 반복)

6) 트립신을 50 mM ABC에 녹여 트립신 대 단백질이 1 대 50 정도의 비율이 되도록 농도를 맞춰 첨가하고,

37℃에서 12 시간 incubation 시켜 펩타이드로 소화시킴.

7) 50 mM ABC 40 μL를 첨가하고, 모든 solution이 필터를 통과할 때까지 centrifuge 함. (2회 반복)

8) Speed-Vac 으로 건조함.

3.2. Desalting

1) Salt를 제거하기 위해 C18 Micro Spin-Column으로 desalting 함.

2) Column 준비

- 80% ACN (in 0.1 % TFA) 75 μL 첨가.

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- 99.9% DIW(in 0.1% TFA) 75 μL 첨가.

- 99.9% DIW, 0.1% TFA 75 μL 첨가.(x2)

3) Sample 준비

- 99.9% DIW(in 0.1% TFA) 75 μL 첨가.

4) 시료를 로딩 시킴.

5) 75 μL의 99.9% DIW(in 0.1% TFA)를 첨가하여 불필요한 물질을 제거함.

6) 펩타이드 용리

- 80% ACN (in 0.1 % TFA) 75 μL 첨가.(x2)

- 100% ACN 75 μL 첨가.

- 80% ACN (in 0.1 % TFA) 75 μL 첨가.

7) Desalting이 끝나면 Speed-vac으로 건조시켜 용액을 모두 제거하고, 분석 전까지 –20℃에 보관함.

3.3. LC-MS/MS 분석

Parameters Conditions

Trapping Column C18, 3 μm, 100 Å, 75 μm x 2 cm

PepMapTM RSLC C18

Analytical Column
2 μm, 100 Å, 75 μm x 50 cm

A: Water with 0.1% formic acid

Mobile Phase
B: 80% ACN with 0.1% formic acid

Time
0 14 75 125 145 147 160 161 185
(min)
Gradient

Solvent
4 4 15 28 40 96 96 4 4
B(%)

Column Flow Rate 300 nL/min

Mass Range 400 ~ 2000 m/z

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3.4. 데이터 분석

1) 데이터 분석을 위해 각 시료 별로 LC-MS/MS 데이터를 Proteome Discoverer를 이용하여 분석함.

2) 데이터베이스는 Uniprot의 Mouse 를 사용함.

3) Modification은 Acetyl, Oxidation, Carbamyl, Carbamidomethyl을 추가함.

4. 연구결과

4.1. LC-MS chromatogram

RT: 0.00 - 185.01 RT: 0.00 - 185.00 RT: 0.00 - 185.01
95.06 NL: 94.70 NL: 94.49 NL:
653.01855 1.91E9 653.01788 1.73E9 653.01801 1.79E9
100 100 100
Base Peak Base Peak Base Peak
MS MS MS
95 95
EBG23PR 95
EBG23PR EBG23PR
156.62 O94_IJU_ O94_IJU_ O94_IJU_
90 664.46021 WT_ND1
90 WT_ND2
90 WT_ND3

85 85 85
67.56
80 80 80 698.70740

75 75 75
116.07
68.06 75.55 895.95355
70 76.19 70 70
698.70874 116.38 698.88214
698.88165 895.95313
65 65 116.10 65
67.67 895.95294
164.73 698.70856 119.57
60 60 119.62 60
620.43811 870.96851
870.97028
Relative Abundance

Relative Abundance

Relative Abundance
55 119.86 55 55
870.96893 75.76
91.63 698.88159
50 50 50 90.65 127.17
748.43518 127.24
46.20 748.43536 812.95117
127.58 812.95001
45 812.95172 45 45 488.72803

47.36 90.98 160.68 42.00

40 83.55 40 42.08 748.43475 129.60 40
488.72824 108.50 620.43695 599.76514 82.46 108.05
650.31488 599.76575 99.25
718.38086 1020.53601 650.31360 718.38202 134.61 164.38
35 35 46.34 473.28000 35 995.02240 664.46295
134.85 488.72769 160.49
30 30 76.59 30 36.69
995.02234 49.24 620.43768
50.54 752.38812 612.81500
644.37225 36.91 132.85 644.37256 142.34
25 24.08 25 612.81494 49.43 823.92041 25 23.48
574.28485 37.65 504.56396 897.05090
644.37286 59.76
612.81519 60.04 142.55
20 20 20 645.33502
619.78748 897.05121 32.62 60.03
537.28314 645.33447 141.07
15 143.85 15 23.54 15
885.91168
729.42487 568.95624
155.66
10 10 155.63 10 621.39349
144.44
685.44934 702.43610
5 5 5
12.69 23.02 166.60 11.16 21.98 167.83 11.22 22.27 180.18
445.12003 445.12003 576.41040 445.12003 445.12003 653.22375 445.12003 445.12003 445.12003
0 0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Time (min) Time (min) Time (min)

WT_ND1 WT_ND2 WT_ND3

RT: 0.00 - 185.00 RT: 0.00 - 185.00 RT: 0.00 - 185.00
94.47 NL: 93.53 NL: 93.34 NL:
653.01794 1.70E9 653.01849 1.98E9 653.01819 1.98E9
100 100 100
Base Peak Base Peak Base Peak
MS MS MS
95 95
EBG23PR 95
EBG23PR EBG23PR
O94_IJU_ 66.09 O94_IJU_ O94_IJU_
90 WT_ND4
90 698.70959 WT_STZ1
90 WT_STZ2
65.92
85 85 85 698.71069
74.53 74.34
698.88177 698.88373
80 80 80

75 67.34 116.03 75 75
698.70837 895.95355
70 70 70
115.40 115.37
65 75.50 65 45.55 895.95392 65
698.88232 488.72775 895.95343
119.44
60 870.96857 60 118.65 60
Relative Abundance

Relative Abundance

Relative Abundance

870.96802
89.97 118.54
55 127.18 55 748.43500 55 870.96948
98.35 45.25
90.57 812.95032 126.34
41.39 473.27957 488.72888 98.09
50 748.43402 50 812.95178 50 89.69 126.27
599.76556 473.27911
748.43481 812.95160
82.20
45 45 650.31329 45
127.35 81.89
46.27 100.39 1036.50879 41.13 650.31293 133.67
40 40 48.72 40
488.72839 129.60 749.91046 599.76544 995.02136
644.37292 48.46 100.19
108.09 1020.53467
35 76.25 35 142.03 35 644.37250 749.90918
39.00 718.38153 142.00
23.51 752.38837 897.05078 897.05048
665.83765 164.66 56.61
568.95526 56.51
30 134.57 620.43713 30 598.28351 30 38.33
49.37 35.66 598.28339
995.02148 142.24 612.81573 596.31287
25 644.37250 25 25
897.05139
24.36
57.28
20 20 20 602.28107
36.27 598.28369 24.45 142.73
692.33807 143.59 602.29004 927.82312 143.43
15 656.98669 15 15 656.98712

10 10 10
143.97 149.51 149.55
685.44934 683.40326 683.40259
5 5 159.91 5 159.79
11.11 22.97 172.23 10.04 21.78 773.49335 165.63 10.07 22.02 773.49341 165.51
445.12003 445.12003 445.12003 445.12003 445.12003 449.28714 445.12003 445.12003 449.28720
0 0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Time (min) Time (min) Time (min)

WT_ND4 WT_STZ1 WT_STZ2

RT: 0.00 - 185.00 RT: 0.00 - 185.01 RT: 0.00 - 185.01
93.37 NL: 93.66 NL: 94.43 NL:
653.01923 2.08E9 653.01953 1.97E9 653.01794 1.79E9
100 100 100
Base Peak Base Peak Base Peak
MS MS MS
95 95
EBG23PR 95
EBG23PR EBG23PR
O94_IJU_ O94_IJU_ O94_IJU_K
90 WT_STZ3
90 66.40 WT_STZ4
90 O_ND1
698.70886
65.93 74.33 74.74
85 698.70959 698.88251 85 698.88208 85

80 80 80
115.93
75 75 75 895.95221
67.52
70 70 70 698.70807
115.63 75.71
65 65 895.95306 65 698.88184
115.50 45.63
119.54
895.95361 488.72787
60 60 60 870.97028
45.43
Relative Abundance

Relative Abundance

Relative Abundance

127.25
488.72806 812.95129
55 55 41.55 90.00 55
89.68 599.76538 748.43512 118.91
50 41.33 118.75 50 870.96954 50 90.47
748.43469 98.07 870.96906 98.35 126.65
599.76569 82.27 748.43500
81.95 473.28061 473.28052 812.95172
45 45 650.31445 45 46.19
650.31427 133.93 108.20
127.56 488.72778
995.02179 48.77 659.31531 129.69
40 48.56 40 1036.51025 142.13 40
644.37280 1020.53717
644.37274 107.57 897.05151 100.90
142.10 718.38092 23.53 749.91083
35 107.32 897.05035 35 35 574.28491 82.03
718.38000 36.68 134.64
36.02 850.44086
612.81482 995.02063 142.27
30 35.62 30 612.81531 56.66 30 49.26
897.05072
612.81543 598.28375 644.37286 164.56
56.56
25 24.30 25 24.23 25 664.46307
598.28363 143.56
574.29004 574.29077 59.69 656.98663
20 20 35.65 143.52 20 33.25 645.33453
31.35 142.80 677.81653
692.33716 656.98706
537.28278 927.82349
15 15 15

10 10 149.62 10 155.77
147.15
815.43683 683.40259 724.45044
5 159.77 5 159.64 5
12.90 22.30 168.58 10.88 23.14 773.49353 168.80 11.63 22.49 172.40
773.49329
445.12003 445.12003 445.12003 445.12003 445.12003 445.12003 445.12003 445.12003 445.12003
0 0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Time (min) Time (min) Time (min)

WT_STZ3 WT_STZ4 KO_ND1

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RT: 0.00 - 185.00 RT: 0.00 - 185.01 RT: 0.00 - 185.00

94.68 NL: 94.46 NL: 94.07 NL:
653.01874 1.47E9 653.01935 1.58E9 653.01782 1.71E9
100 100 100
Base Peak Base Peak Base Peak
MS MS MS
95 95
EBG23PR 95
EBG23PR EBG23PR
O94_IJU_K O94_IJU_K O94_IJU_K
90 O_ND2
90 O_ND3
90 O_ND4

85 85 85
115.98
80 80 895.95294 80

115.79
75 75 75
66.97 895.95258
70 70 70 698.70825
67.21
65 65 698.70807 65 75.08
119.43
67.58 115.99 698.88141
870.96967
60 698.70874 895.95361 60 75.39 60
127.08
Relative Abundance

Relative Abundance

Relative Abundance
698.88239 812.95154 119.25
55 55 55 870.96887
75.90 119.47 89.87
870.96857 126.94
50 698.88214 50 50 748.43488 812.95172
90.42 129.52
45.51
127.27 748.43591 1020.53589 488.72772
45 45 45
812.95197 46.09
90.96 100.79
40 46.37 748.43500 40 488.72784 749.91138 142.19 40 129.31
488.72849 108.25 897.05145 41.35 1020.53778
129.73 36.54
35 659.31458 35 35 599.76550 75.88 134.30
1020.53577 612.81543 107.60
155.72 752.38867 718.38202 995.02271 155.70
23.48 76.23
76.72 99.22 574.28552 752.38867 702.43420 702.43237
30 30 30 142.09
37.04 752.38947 473.27991 164.52
134.62 664.46362 49.05 23.41 35.73 48.56 897.05060
612.81561 83.83
25 23.65 49.44 581.31323 995.02081 25 644.37262 25 568.95575 612.81506 644.37250
143.38 164.48
568.95544 644.37262 59.61
142.22 155.74 729.42511 664.46289 59.33
20 20 645.33362 20 31.40 645.33362 164.49
57.27 897.54889 724.44684 34.33 155.35 143.41 664.46301
598.28351 909.46606 614.38489 537.28320 656.98669
32.65 143.51
15 15 15
537.28308 656.98676
10 10 10 155.28
155.37 614.38336
614.38458
5 5 5
12.51 22.30 173.00 11.60 22.66 172.74 11.79 23.03 172.39
445.12003 445.12003 445.12003 445.12003 445.12003 445.12003 445.12003 445.12003 445.12003
0 0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Time (min) Time (min) Time (min)

KO_ND2 KO_ND3 KO_ND4

RT: 0.00 - 185.00 RT: 0.00 - 185.00 RT: 0.00 - 185.01
93.82 NL: 93.64 NL: 95.43 NL:
653.01837 1.75E9 653.01904 1.97E9 653.01910 9.86E8
100 100 100
66.64 Base Peak Base Peak Base Peak
698.70764 MS MS MS
95 95
EBG23PR 95
EBG23PR EBG23PR
75.17 O94_IJU_K O94_IJU_K O94_IJU_K
90 698.88208 O_STZ1
90 O_STZ2
90 O_STZ3

85 85 66.36 85
698.70868
74.73
80 80 80
698.88239
115.69
75 895.95349 75 75
115.42
70 70 895.95044 70 67.88
698.70905
65 65 65
46.24 76.53
60 488.72818 60 60 698.88239
45.63 118.74
Relative Abundance

Relative Abundance

Relative Abundance
118.90 870.96967
90.36 488.72891
55 870.96930 55 89.99 55
748.43481 98.65
126.56 748.43512 42.92 117.87
473.27972
50 82.71 812.95135 50 50 599.76563 895.95331
126.47
650.31476 98.29 812.95135
134.04
45 45 473.28064 45
995.02100
82.28 100.35 129.41
40 49.31 107.66 40 650.31445 749.91046 1020.53638 40
48.79 120.96
644.37256 718.38141
644.37280 91.86 870.97058
35 142.11 35 107.58 142.05 35 748.43524 100.34
37.76 897.05103 659.31458 897.05096 473.27982 128.59
481.73767 59.39 35.98 812.95123
30 30 612.81598 56.83 30
645.33350 37.19
598.28375 80.24
612.81555 130.86
25 24.39 25 25 545.79865
58.04 720.39935
504.57202
20 142.75 20 24.25 20 36.84 598.28369
36.36 602.28052 142.73 143.46
927.82300 927.82355 692.33673
692.33728 897.05005
15 15 15 25.18
143.95 602.28485 144.76
10 149.54 10 1009.85944 10 656.98700 160.68
683.40277 154.67 773.49408
5 159.71 5 5
11.78 21.92 773.49359 168.66 11.12 21.82 1000.00739 164.05 182.55 7.97 24.89 165.76
445.12003 445.12003 445.12003 445.12003 445.12003 840.56781 445.12003 445.12003 574.26550 625.39215
0 0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Time (min) Time (min) Time (min)

KO_STZ1 KO_STZ2 KO_STZ3

4.2. 데이터 분석

5. 참조자료의 [별첨1] 문서 참조

5. 참조자료

[별첨1] 인제대학교-한진_Data analysis_20231020

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ExDEGA Column Name Description

Accession 단백질 고유의 ID

Description 단백질 설명

Fold change Control과 Test의 Abundance Ratio (Test/Control)

p-value Abundance Ratio P-Value

Average of normalized data (log2) Log2(Average of normalized data)

Normalized data (log2) Log2(Normalized value using Total Peptide Amount)

Raw Data Abundance value

Function 단백질 기능

Coverage [%] 단백질 전체 A.A sequence 중에서 매칭된 A.A의 %

Peptides 단백질 동정에 사용된 펩타이드의 수

PSMs

(Number of peptide spectrum 펩타이드에 매칭된 스펙트럼 수

matches)

단백질 동정에 사용된 펩타이드 중, 전체 DB에서 고유한

Unique peptides
sequence를 갖는 펩타이드의 수

AAs 단백질 A.A 개수

MW [kDa] 단백질 분자량

calc.pI 단백질 pI 값