Development of a liquid chromatography tandem mass spectrometry method to
investigate the presence of biomarkers of DNA damage in urine related to red
meat consumption and risk of colorectal cancer
Chiara Da Pieve1*, Natasha Sahgal1, Sharon A. Moore2, Maria N. Velasco-Garcia1*
1
Life, Health and Chemical Sciences, The Open University, Milton Keynes, MK7
6AA, UK
2
School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University,
Liverpool, L3 3AF, UK
* Correspondence to: C. Da Pieve and M. Velasco-Garcia, Department of Life, Health
and Chemical Sciences, Faculty of Science, The Open University, Walton Hall,
Milton Keynes, UK, MK7 6AA. Phone: +44 1908652515, Fax: +44 1908858327. Email: m.n.velasco@open.ac.uk, chiara.dapieve@icr.ac.uk
Contents:
SRM transitions for O6MeG, O6MedG and Tubercidin
Page 2
ESI full scan and product ion spectra of tubercidin (ISTD)
Page 3
Calibration curves for O6CMG and O6CMdG
Page 4
Mass chromatograms of clinical urine samples
Pages 5-9
Calibration curves for O6MeG and O6MedG
Page 10
Mass chromatogram of depurination sample using the acetonitrile-FA
Page 11
elution gradient
1
SRM transitions for O6MeG, O6MedG and Tubercidin
Adduct
Precursor Ion
(m/z)
Product Ions
(m/z)
Collision
Energy (eV)
O6MeG
166.2
67.5
134.2
149.2
32
23
19
O6MedG
282.2
134.2
149.3
166.2
36
34
13
267.2
108.4
118.2
135.2
43
41
21
Tubercidin
(ISTD)
Table S1. SRM transitions for O6MeG, O6MedG and Tubercidin. The quantitation
transition is underlined.
2
ESI full scan and product ion spectra of tubercidin
Figure S1. ESI full scan (top) and product ion (bottom) spectra of tubercidin (ISTD).
The product ions scan spectrum was achieved by collision-induced dissociation of the
m/z 267.2 ([M+H]+) using a CE of 40 V.
3
Calibration curves for O6CMG and O6CMdG
6
3.0 O CMG
Y = -0.03923+0.02806*X
2
r = 0.998
Area Ratio
2.5
2.0
1.5
1.0
0.5
0.0
0
20
40
60
80
100
ng/mL
6
O CMdG
7
Y = -0.10474+0.04844*X
2
r = 0.999
6
Area Ratio
5
4
3
2
1
0
0
20
40
60
80
100
120
140
160
ng/mL
Figure S2. Calibration lines, conducted in triplicate, for the determination of O6CMG
(top) and O6CMdG (bottom) in synthetic urine by SRM. Concentration ranging
between 0 and 100 ng/mL, and 0 and 140 ng/mL were used for O6CMG and O6CMdG
respectively.
4
Mass chromatograms of clinical urine samples
RT: 3.12 - 5.15
RT: 5.15 NL:
AA: 14346 1.51E3
SN: 87
TIC MS
251109AD
S6C
(a)
1500
1450
5.11
1400
1350
1300
1250
1200
1150
1100
1050
1000
950
900
RT: 3.38
MA: 2388
SN: 57
Intensity
850
800
750
700
RT: 3.53
MA: 697
SN: 23 RT: 3.61
MA: 2021
SN: 23
3.67 3.71
650
600
550
500
RT: 3.89
MA: 293
SN: 14
450
RT: 4.17
AA: 1136
SN: 18
RT: 3.99
AA: 605
SN: 12
RT: 4.56
AA: 932 RT: 4.65
SN: 16 AA: 852
SN: 11
RT: 4.32
AA: 1806
SN: 19
4.20
350
4.92 4.94
4.74
400
4.79
4.83
3.95
4.07
3.79
300
4.12
4.49
250
200
150
100
50
3.17 3.23
0
C:\Xcalibur\...\Urinesamples\251109ADS6C
3.2
3.3
RT: 9.00 - 11.50
RT: 9.01
AA: 462
SN: 9
3.4
11/26/2009 15:06:09
3.5
3.6
3.7
3.8
3.9
4.0
4.1
4.2
Time (min)
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.0
5.1
NL:
3.94E2
(b)
TIC MS
251109AD
S6C
380
360
RT: 9.21
AA: 338
SN: 13
340
320
300
RT: 9.11
MA: 318
SN: 11
280
RT: 9.97
MA: 1357
SN: 19RMS
260
RT: 9.49
MA: 247
SN: 11RMS
240
Intensity
220
9.87
200
9.41
180
9.66
160
9.27
9.82
9.38
9.55
140
120
9.74
100
80
60
40
10.07
20
10.26
10.16
10.67
11.25
10.95
10.76
10.34 10.44
10.51
11.13 11.16
10.86
11.41
0
9.0
9.2
9.4
9.6
9.8
10.0
10.2
Time (min)
10.4
10.6
10.8
11.0
11.2
11.4
Figure S3. LC-MS/MS SRM chromatogram after direct injection of 24h urine sample
of a volunteer after red meat diet. Each chromatogram is divided in two time windows
corresponding to: (a) O6CMG; (b) O6CMdG.
5
RT: 3.01 - 5.13
NL:
4.47E2
(a)
TIC MS
251109AD
S3A
560
540
520
500
480
RT: 3.76
MA: 1176
SN: 21
460
440
RT: 3.88
MA: 590
SN: 20
420
RT: 3.54
MA: 658
RT: 3.62
SN: 14
MA: 364
3.71
SN: 14
3.48
400
RT: 3.34
3.39
MA: 1194
SN: 23
380
360
340
RT: 4.88
MA: 974
SN: 18
4.79
4.72
RT: 4.06
MA: 741
SN: 15
320
Intensity
RT: 4.76
MA: 738
SN: 12
RT: 4.54 4.58
MA: 996
SN: 13
300
RT: 4.39
MA: 310
SN: 12
280
260
RT: 4.16
MA: 933
SN: 10 4.20
240
220
4.51
4.63
4.93
4.26
4.01
200
3.95
5.06
180
160
140
120
100
80
60
40
3.07 3.13
20
3.20
0
3.1
3.2
3.3
3.4
C:\Xcalibur\...\Urinesamples\251109ADS3A
3.611/26/2009
3.7 00:38:20
3.8
3.5
3.9
4.0
4.1
Time (min)
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.0
5.1
RT: 8.99 - 11.50
(b)
NL:
4.63E2
TIC MS
251109AD
S3A
460
440
420
400
RT: 9.33
AA: 616
SN: 15
380
360
340
320
300
280
Intensity
260
9.07
RT: 9.70
AA: 515
SN: 10
240
220
9.18
9.27
9.15
200
RT: 9.88
RT: 9.98
MA: 233
MA: 292
SN: 10
SN: 10
9.75
RT: 9.57
MA: 202
SN: 7RMS
180
160
9.41
140
9.96
120
9.46
100
80
60
10.49 10.57
40
10.14
10.29
10.74
10.86 10.93
10.42
11.04
11.14 11.23
20
11.30
11.37
0
9.0
9.2
9.4
9.6
9.8
10.0
10.2
Time (min)
10.4
10.6
10.8
11.0
11.2
11.4
Figure S4 LC-MS/MS SRM chromatogram after direct injection of 24h urine sample
of a volunteer after vegetarian diet. Each chromatogram is divided in two time
windows corresponding to: (a) O6CMG; (b) O6CMdG.
6
RT: 2.81 - 5.28
NL:
3.77E2
(a)
TIC MS
291111_A
DS3_30-6
400
5.27
380
360
340
320
300
RT: 3.13
MA: 1093
SN: 16
280
RT: 3.37
MA: 215
SN: 9
260
3.10
Intensity
240
RT: 3.53
MA: 345
SN: 10
3.22
RT: 4.16
AA: 799
SN: 15
RT: 3.65
MA: 988
SN: 11
RT: 3.91
MA: 300
SN: 8
220
RT: 4.51
MA: 495
SN: 13
RT: 4.33
AA: 317
SN: 15
5.19
RT: 4.79
MA: 371
SN: 10
4.20
3.71
RT: 4.91
AA: 214
RT: 5.00
SN: 10
AA: 192
SN: 9
200
3.32
180
3.45
4.08
4.27
160
5.07
140
3.96
120
4.42
4.59
4.67
3.85
100
80
60
40
2.96
20
2.89
0
C:\Xcalibur\...\291111\291111_ADS3_30-6
2.9
3.0
3.1
3.2
3.3
11/29/2011
3.5
3.618:20:34
3.7
3.4
3.8
3.9
4.0
4.1
Time (min)
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.0
5.1
5.2
RT: 9.00 - 11.50
80
NL:
8.05E1
RT: 10.21
MA: 146
SN: 4
(b)
75
TIC MS
291111_A
DS3_30-6
RT: 9.94
MA: 107
SN: 4
70
65
60
RT: 9.83
MA: 81.52
SN: 3
55
50
RT: 9.24
MA: 48.45
SN: 2
Intensity
45
40
RT: 11.19
MA: 93.08
SN: 2RMS
RT: 9.46
MA: 42.96
SN: 1RMS
11.15
9.10
35
11.46
10.30
11.03
10.98
10.51
30
9.30
9.78
11.40
10.79
10.00 10.12
25
9.74
9.42
20
9.57
9.02
10.88
10.41
10.56
11.25
10.75
9.65
11.29
10.70
10.62
15
10
5
0
9.0
9.2
9.4
9.6
9.8
10.0
10.2
Time (min)
10.4
10.6
10.8
11.0
11.2
11.4
Figure S5. LC-MS/MS SRM chromatogram after C18-SPE purification of 24h urine
sample of volunteer after red meat diet. Each chromatogram is divided in two time
windows corresponding to: (a) O6CMG; (b) O6CMdG.
7
RT: 2.91 - 5.22 SM: 15G
(a)
RT: 4.11
MA: 1089
SN: 11
640
NL:
1.37E3
4.38
TIC MS
071111_A
DS2_28-6
620
600
580
RT: 3.94
AA: 1078
SN: 83
RT: 3.16
MA: 2840
3.18
SN: 18
560
540
RT: 4.77
MA: 662
SN: 5
4.51
520
RT: 4.92
MA: 246
SN: 3
500
480
RT: 3.75
MA: 425
SN: 5
460
440
3.55
420
3.64
400
380
3.31
360
Intensity
340
320
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
2.95
0
C:\Xcalibur\...\071111\071111_ADS2_28-6
3.0
3.1
3.2
3.3
11/7/2011 20:26:46
3.4
3.5
3.6
3.7
3.8
3.9
RT: 8.99 - 11.49
4.0
4.1
Time (min)
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.0
5.1
5.2
NL:
1.39E2
(b)
145
TIC MS
071111_A
DS2_28-6
RT: 10.99
MA: 186
SN: 5
140
135
130
125
120
115
110
RT: 9.40
MA: 153
SN: 4
105
100
95
90
Intensity
85
RT: 9.13
MA: 156
SN: 3
80
RT: 9.26
MA: 149
SN: 3
11.46
RT: 10.57
MA: 187
SN: 3
75
70
11.23
65
11.04
RT: 10.02
MA: 74.41
SN: 2
60
55
11.08
10.68
50
45
11.28
11.13
10.78
40
35
10.33 10.42
9.47
9.55
25
10.87
10.22
30
9.88
9.64
9.67
9.71
20
9.84
10.45
10.12
9.98
15
10
5
0
9.0
9.2
9.4
9.6
9.8
10.0
10.2
Time (min)
10.4
10.6
10.8
11.0
11.2
11.4
Figure S6. LC-MS/MS SRM chromatogram after C18-SPE purification of 24h urine
sample of volunteer after vegetarian diet. Each chromatogram is divided in two time
windows corresponding to: (a) O6CMG; (b) O6CMdG.
8
RT: 2.99 - 5.07
(a)
NL:
4.34E2
TIC MS
ICIS
120312_A
DS3_286_wash1
RT: 3.25
AA: 1477
SN: 87
310
300
290
280
270
260
250
240
230
220
210
200
190
Intensity
180
170
160
150
140
RT: 3.43
AA: 304
SN: 29
130
120
110
100
RT: 3.78
AA: 155
SN: 15
90
80
RT: 4.30
AA: 148
SN: 17
RT: 4.05
AA: 173
SN: 14
70
60
50
40
30
20
10
0
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.0
4.1
Time (min)
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.0
RT: 2.89 - 5.05
NL:
3.71E2
(b)
TIC MS
ICIS
120312_A
DS3_195_wash1
180
170
160
150
140
130
120
Intensity
110
100
90
80
70
RT: 4.03
RT: 3.68
60
RT: 3.38
RT: 3.87
50
RT: 4.40
RT: 3.53
RT: 4.21
40
30
20
10
0
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.0
Time (min)
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.0
Figure S7. LC-MS/MS SRM chromatogram after acidic hydrolysis and C18-SPE
purification of 24h urine sample of volunteer after either the red meat (a) or the
vegetarian (b) diet. The transition corresponding to O6CMG is shown.
9
Calibration curves for O6MeG and O6MedG
35
6
O MeG
30
Y = -0.05231+0.45766*X
2
r = 0.998
Area Ratio
25
20
15
10
5
0
0
10
20
30
40
50
60
70
80
70
80
ng/mL
5
6
O MedG
Y = 0.00608+0.05956*X
2
r = 0.998
Area Ratio
4
3
2
1
0
0
10
20
30
40
50
60
ng/mL
Figure S8. Calibration lines, conducted in triplicate, for the determination of O6MeG
(top) and O6MedG (bottom) in synthetic urine by SRM. Concentration ranging
between 0 and 70 ng/mL were used for O6MeG and O6MedG.
10
Mass chromatogram of depurination samples using the acetonitrile-FA elution
gradient
Figure S9 LC-MS/MS SRM chromatogram after acidic hydrolysis and C18-SPE
purification of synthetic urine samples (N=3) spiked with 20 ng/mL of O6CMdG. The
peak eluting at 7.2 min. is the ISTD (2.2ng/mL).
11