Identification and Quantification of Metabolic Modification of Serum Protein as a Prognostic Biomarker by Liquid Chromatography-Mass Spectrometry

(1)

Identification and Quantification of

Metabolic Modification of Serum

Protein as a Prognostic Biomarker by

Liquid Chromatography-Mass

Spectrometry

Speaker：Hsin Chen

Advisor：Shu-Hui Chen

(2)

(3)

R

HO

E1:

E,

=

O

E2:

R,

-

OH

P450

enzymes

R

HO

OH

R

HO

2 -

_OHE1(E2)

4 -

_OHE1(E2)

peroxidase

or

_P450

peroxidase

or

_P450

R

O

E1(E2)

-

2,3

-

_Q

R

O

E1(E2)

-

3,4

-

_Q

DNA

stable

adducts

stable

adducts

COMT

R

HO

H

₃

CO

2 -

OCH

₃

E1(E2)

R

HO

OCH

₃

4 -

OCH

₃

_E1(E2)

Cavalieri, Ercole, et al. "Catechol estrogen quinones as initiators of breast and other

human cancers: implications for biomarkers of susceptibility and cancer prevention."

Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1766.1 (2006): 63-78.

catechol estrogens (CEs)

Toxic intermediate

cancer

Estrogen Hormone – Metabolism pathway

conjugate with Cysteine,

Histidine and Lysine.

Biomarkers

(4)

Sample

Inlet

Ion

Source

Analyzer

Mass

Ion

Detector

Data

output

Vacuum Pump

column

HPLC system

liquid chromatography-mass spectrometry

(5)

Mass analyzer

LTQ-Orbitrap

4 Fourier transform

(6)

Estrogenized serum proteins identified from diabetic

patients by bottom-up proteomics

(7)

Human Serum Albumin (HSA)

 35~50mg/mL in serum

 MW.：66.4kDa

 balance pH and osmotic pressure



The most abound protein in the serum

 contain 35 cysteines, C34 is a free thiol group.

(8)

A L V L I A

F

A

Q

Y

L

Q

C

P F E D H V K

Alkylation

A L V L I A

F

A

Q

Y

L

Q

C

P F E D H V K

Estrogenization

7 1. Dithiothreito

2. iodoacetamide

=

+

(9)

Identification of estrogenization of serum proteins

in diabetic patients by LC/MSMS

(10)

Analysis Process

(11)

Solid phase extraction

(12)

Multiple Reaction Monitoring (MRM)

 Three product ions for one peptide

(13)

20161222_0212_noc34_nospe_1 #7890-7954RT:40.37-40.65AV:16NL:2.91E2

F:ITMS + c NSI SRM ms2 [email protected] [1068.01-1072.01, 1103.53-1107.53, 1160.07-1164.07]

1069 1070 1071 1072 m/z 1104 1105 1106 1107 m/z 1161 1162 1163 1164 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1069.32 1071.19 1070.39 1068.20 1104.84 1105.91 1104.04 1107.50 1161.42 1161.90 1162.64 1163.57 1160.27

MS2 Fragment Spectrum of alkylated

C

34 Peptide Ions

(14)

20161222_0212_noc34_nospe_1 #7890-7954RT:40.37-40.65AV:16NL:2.91E2

1069 1070 1071 1072 m/z 1104 1105 1106 1107 m/z 1161 1162 1163 1164 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1069.32 1071.19 1070.39 1068.20 1104.84 1105.91 1104.04 1107.50 1161.42 1161.90 1162.64 1163.57 1160.27

MS2 Fragment Spectrum of Estrogenized

C

34 Peptide Ions

(15)

15 RT:

0.00 - 70.00

0

10

20

30

40

50

60 Time (min)

0

20

40

60

80

100

0

20

40

60

80

100 R

el

at

iv

e A

bundanc

e

20160830_160525_200ugst+50ng4ohe2_80%spe_800nl #8882-9034RT:43.38-44.08AV:39NL:3.61E4

F:FTMS + p NSI Full ms [300.00-2000.00] 904.5 905.0 905.5 906.0 906.5 907.0 907.5 908.0 908.5 909.0 909.5 910.0 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 906.81 z=3 907.14 z=3 906.47 z=3 907.48 z=3 907.81 z=3 904.42 z=2 908.02 z=? 908.52z=? 904.53 z=? 905.44 z=? 904.92 z=2 909.02z=? 909.45z=? 906.18 z=? 905.85 z=? 909.97z=?

20160829_160822_200ugo4_500nl #8890-9049RT:43.38-44.06AV:40NL:3.97E4

F:FTMS + p NSI Full ms [300.00-2000.00] 904.5 905.0 905.5 906.0 906.5 907.0 907.5 908.0 908.5 909.0 909.5 910.0 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 904.31 z=8 904.44 z=8 905.79 z=? 905.57 z=? 906.08 z=? 905.46 z=? 906.50 z=? 904.77 z=? 904.49 z=? 909.75 z=? 909.45 z=? 906.83 z=? 904.97 z=? 905.22 z=? 909.82 z=? 908.82 z=? 908.47 z=? 908.02 z=? 906.98 z=? 907.48z=? 909.08z=? 909.88z=?

serum

Activated serum

Limit of quantification.(LOQ)

S/N ratio <3

S/N = 28

(16)

without SPE

_{with SPE}

20160730_160726_200ugO17_80%SPE_800nL

#

6170-6359

RT:

42.08-43.16

AV:

48 NL:

8.98E-1

F:

ITMS + c NSI SRM ms2 [email protected] [1068.01-1072.01, 1103.53-1107.53, 1160.07-1164.07]

1069 1070 1071 1072

m/z

1104 1105 1106 1107

m/z

1161 1162 1163 1164

m/z

0

10

20

30

40

50

60

70

80

90

100 R

el

at

iv

e

A

bundanc

e

0

10

20

30

40

50

60

70

80

90

100 R

el

at

iv

e

A

bundanc

e

0

10

20

30

40

50

60

70

80

90

100 R

el

at

iv

e

A

bundanc

e

1069.62

1071.84

1070.71

1068.56

1105.08

1106.17

1103.70

1107.24

1161.63

1163.05

1161.00

1163.91

20160826_160815_200ugO17_500nL

#

9056-9171

RT:

43.51-44.00

AV:

29 NL:

5.70E1

F:

ITMS + c NSI SRM ms2 [email protected] [1068.01-1072.01, 1103.53-1107.53, 1160.07-1164.07]

1069 1070 1071 1072

m/z

1104 1105 1106 1107

m/z

1161 1162 1163 1164

m/z

0

10

20

30

40

50

60

70

80

90

100 R

e

la

ti

v

e A

bundanc

e

0

10

20

30

40

50

60

70

80

90

100 R

e

la

ti

v

e A

bundanc

e

0

10

20

30

40

50

60

70

80

90

100 R

e

la

ti

v

e A

bundanc

e

1071.77

1068.48

1069.68

_1070.65

1104.63

1107.29

1106.26

1103.79

1105.18

1161.10

1162.21

1163.33

20160805_160726_200ugn60_80%spe_800nl #8676-8785RT:42.60-43.06AV:27NL:1.62

1069 1070 1071 1072 m/z 1104 1105 1106 1107 m/z 1161 1162 1163 1164 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1071.67 1068.33 1070.83 1105.25 1104.64 1107.25 1103.75 1106.39 1161.40 1162.40 1160.65 20160826_160815_200ugn60 #9026-9132RT:43.57-44.02AV:27NL:7.66

1069 1070 1071 1072 m/z 1104 1105 1106 1107 m/z 1161 1162 1163 1164 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1072.00 1069.64 1068.19 1104.55 1105.15 1106.06 1104.05 1107.15 1161.16 1163.70 1162.96

16 Background interferences

(17)

y = 0.3166x + 0.0001

R² = 0.9569

0.00%

0.02%

0.04%

0.06%

0.08%

0.10%

0.12%

0.000% 0.100% 0.200% 0.300% 0.400%

inte

ns

ity

Quantity of Added standard to sample

Standard Addition Method

A

B C

Unknown / activated(SPIKE)

Reading of unknown

without added standard

(18)

Positive (activated) serum standard

24hr, 37℃

18 ER(%)=0.33

RT:

0.00 - 70.01

0

5

10

15

20

25

30

35

40

45

50

55

60

65 Time (min)

0

20

40

60

80

100

0

20

40

60

80

100 R

el

at

iv

e A

bundanc

e

Modified

Unmodified

Modified

Estrogenied

modification ratio

(ER%)

=

Area of Estrogenization C34

(19)

No activated

SPIKE 0.19%

SPIKE 0.33%

20160829_160822_200ugO4_500nL #8779-8925 RT:42.90-43.52AV:37NL:1.77

1069 1070 1071 1072 m/z 1104 1105 1106 1107 m/z 1161 1162 1163 1164 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1071.83 1071.18 1105.36 1106.57 1104.10 1107.20 20170113_04_nospe_spike10_4ohe2 #8021-8061RT:40.38-40.55AV:10NL:6.60

1069 1070 1071 1072 m/z 1104 1105 1106 1107 m/z 1161 1162 1163 1164 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1071.9370 1104.9116 1106.6243 1105.7733 1107.3120 1161.5677

20170113_04_nospe_spike20_4ohe2 #8005-8045RT:40.40-40.56AV:10NL:2.37E2

1069 1070 1071 1072 m/z 1104 1105 1106 1107 m/z 1161 1162 1163 1164 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1069.4836 1070.4838 1071.5139 1104.9594 1107.4264 1105.5893 1161.5254 1162.6308_1163.8541