Background
Methods
Patient sample selection
FTICR-MS discovery | MRM validation | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Genomics Collaborative | Seracare 1 | Osaka | Chiba | Seracare 2 | ||||||
CRC | Control | CRC | Control | CRC | Control | CRC | Control | CRC | Control | |
Total
| 40 | 50 | 26 | 25 | 46 | 35 | 40 | 40 | 70 | 70 |
Male
N
| 19 | 24 | 17 | 16 | 27 | - | 19 | 24 | 44 | 41 |
Male age
| 59 (30-78) | 56 (30-78) | 62 (46-80) | 51 (35-70) | 63 (28-90) | - | 68 (45-91) | 48 (36-69) | 67 (39-87) | 63 (32-82) |
Male BMI
| 20.9 ± 3.8 | 25.0 ± 0.9 | 24.3 ± 5.7 | 25.6 ± 4.6 | NA | - | NA | NA | 28.0 ± 4.8 | 26. ± 4.2 |
Female N
| 21 | 26 | 9 | 9 | 19 | - | 21 | 16 | 26 | 29 |
Female age
| 54 (40-82) | 55 (40-79) | 78 (59-86) | 55 (26-95) | 65 (31-77) | - | 70 (51-84) | 49 (39-59) | 73 (35-90) | 56 (26-86) |
Female BMI
| 19.9 ± 4.6 | 24.8 ± 2.2 | 23 ± 3.2 | 29 ± 8.0 | NA | - | NA | NA | 25.5 ± 4.4 | 24.0 ± 4.5 |
Stage 0/I
| 8 | - | 5 | - | 10 | - | 9 | - | 13 | - |
Stage II
| 16 | - | 8 | - | 14 | - | 18 | - | 21 | - |
Stage III
| 15 | - | 8 | - | 12 | - | 11 | - | 25 | - |
Stage IV
| 1 | - | 2 | - | 8 | - | 2 | - | 7 | - |
Unknown
| 0 | - | 3 | - | 2 | - | 0 | - | 4 | - |
Sample extraction
FTICR-MS analysis
Full-scan quadruple time-of-flight (Q-TOF) and high performance liquid chromatography (HPLC)-coupled MS/MS spectrometry
Preliminary isolation of CRC biomarkers and NMR analysis
TQ-MRM methodology
Statistical analysis
Results
FTICR metabolomic profiling
Genomics Collaborative (P< 0.05) | Seracare (P< 0.05) | Osaka (P< 0.05) | |
---|---|---|---|
GCI (Top 50)
| - | 46 (92%) | 31 (62%) |
Seracare 1 (Top 50)
| 35 (70%) | - | 27 (54%) |
Osaka (Top 50)
| 44 (88%) | 47 (94%) | - |
GCI | ||||||
---|---|---|---|---|---|---|
Rank order | Detected mass | Molecular formula | Part per million | Analysis mode |
Pvalue | Ratio (CRC/normal) |
6 | 446.3406 | C28H46O4 | 2.22 | NAPCI | 6.4E-13 | 0.31 |
13 | 448.3563 | C28H48O4 | 2.32 | NAPCI | 2.5E-12 | 0.41 |
8 | 466.3661 | C28H50O5 | 0.59 | NAPCI | 9.4E-13 | 0.25 |
7 | 468.3840 | C28H52O5 | 5.39 | NAPCI | 9.0E-13 | 0.27 |
21 | 492.3829 | C30H52O5 | 2.89 | NAPCI | 8.5E-11 | 0.33 |
24 | 494.3977 | C30H54O5 | 1.16 | NAPCI | 1.9E-10 | 0.35 |
29 | 518.3976 | C32H54O5 | 0.92 | NAPCI | 1.6E-09 | 0.37 |
12 | 538.4259 | C32H58O6 | 4.76 | NAPCI | 2.5E-12 | 0.30 |
44 | 574.4607 | C36H62O5 | 1.7 | NAPCI | 1.6E-08 | 0.40 |
26 | 576.4771 | C36H64O5 | 2.99 | NAPCI | 3.0E-10 | 0.37 |
32 | 578.4931 | C36H66O5 | 3.59 | NAPCI | 3.2E-09 | 0.34 |
11 | 592.4711 | C36H64O6 | 1.37 | NAPCI | 2.2E-12 | 0.27 |
15 | 594.4851 | C36H66O6 | 1.41 | NAPCI | 6.3E-12 | 0.26 |
Seracare
| ||||||
45 | 446.3413 | C28H46O4 | 3.79 | NAPCI | 1.8E-06 | 0.36 |
9 | 448.3570 | C28H48O4 | 3.88 | NAPCI | 1.6E-08 | 0.36 |
3 | 466.3664 | C28H50O5 | 1.23 | NAPCI | 8.5E-10 | 0.34 |
6 | 468.3847 | C28H52O5 | 6.89 | NAPCI | 4.9E-09 | 0.36 |
17 | 492.3835 | C30H52O5 | 4.11 | NAPCI | 4.6E-08 | 0.42 |
34 | 494.3971 | C30H54O5 | 0.05 | NAPCI | 6.6E-07 | 0.41 |
11 | 518.3968 | C32H54O5 | 0.63 | NAPCI | 2.2E-08 | 0.33 |
18 | 538.4263 | C32H58O6 | 5.5 | NAPCI | 7.8E-08 | 0.38 |
32 | 574.4595 | C36H62O5 | 0.39 | NAPCI | 6.1E-07 | 0.32 |
42 | 576.4768 | C36H64O5 | 2.47 | NAPCI | 1.0E-06 | 0.37 |
49 | 578.4933 | C36H66O5 | 3.93 | NAPCI | 3.2E-06 | 0.42 |
30 | 592.4721 | C36H64O6 | 3.06 | NAPCI | 5.6E-07 | 0.27 |
50 | 594.4851 | C36H66O6 | 1.41 | NAPCI | 3.7E-06 | 0.32 |
Osaka
| ||||||
6 | 446.3400 | C28H46O4 | 0.87 | NESI | 1.8E-10 | 0.44 |
13 | 448.3556 | C28H48O4 | 0.76 | NESI | 2.2E-09 | 0.54 |
1 | 466.3663 | C28H50O5 | 1.02 | NESI | 2.9E-12 | 0.50 |
5 | 468.3815 | C28H52O5 | 0.05 | NESI | 1.8E-10 | 0.49 |
4 | 492.3814 | C30H52O5 | 0.15 | NESI | 7.1E-11 | 0.57 |
23 | 494.3969 | C30H54O5 | 0.45 | NESI | 2.0E-07 | 0.62 |
39 | 518.3975 | C32H54O5 | 0.72 | NAPCI | 5.8E-06 | 0.52 |
19 | 538.4237 | C32H58O6 | 0.67 | NESI | 4.7E-08 | 0.58 |
16 | 574.4600 | C36H62O5 | 0.48 | NESI | 3.8E-09 | 0.42 |
7 | 576.4756 | C36H64O5 | 0.39 | NESI | 3.0E-10 | 0.42 |
14 | 578.4910 | C36H66O5 | 0.04 | NESI | 2.6E-09 | 0.50 |
15 | 592.4703 | C36H64O6 | 0.02 | NESI | 3.3E-09 | 0.41 |
3 | 594.4859 | C36H66O6 | 0.07 | NESI | 6.8E-11 | 0.40 |
Structural elucidation
Marker nominal neutral mass | 446 | 448 | 450 | 464 | 466 | 468 | |
---|---|---|---|---|---|---|---|
[M-H]-(%) | 445 (100%) | 447 (52%) | 449 (92%) | 463 (70%) | 465 (100%) | 467 (100%) | |
Chain cut ions (%)
|
223 (18%) |
277 (11%) |
171 (7%) |
277 (10%) |
241 (7%) |
187 (12%) | |
222 (11%) |
239 (5%) |
127 (9%) |
241 (68%) |
223 (3%) |
169 (3%) | ||
207 (3%) |
207 (3%) |
125 (12%) |
223 (15%) |
215 (2%) |
141 (2%) | ||
205 (11%) |
169 (6%) |
113 (38%) |
185 (8%) |
185 (4%) |
113 (4%) | ||
113 (5%) |
113 (25%) |
167 (4%) |
167 (4%) | ||||
113 (28%) |
113 (7%) | ||||||
Peripheral cut ions (%) |
Loss of H
2
O
|
427 (50%) |
429 (35%) |
431 (80%) |
445 (46%) |
447 (45%) |
449 (84%) |
Loss of 2H
2
O
|
409 (8%) |
411 (6%) |
413 (13%) |
427 (6%) |
429 (8%) |
431 (10%) | |
Loss of CO
2
|
401 (95%) |
403 (100%) |
405 (100%) |
419 (100%) |
421 (45%) |
423 (25%) | |
Loss of CO
2
and H
2
O
|
383 (28%) |
385 (15%) |
387 (32%) |
401 (24%) |
403 (20%) |
405 (13%) | |
*Loss of CO
2
and 2H
2
O
|
365
|
367
|
369
|
383 (2%) |
385 (4%) |
387 (3%) | |
*Loss of 3H
2
O
|
409
|
411
|
413
| ||||
Secondary daughter ions (%)
|
357 (5%) |
331 (3%) |
307 (5%) |
347 (5%) |
349 (4%) |
349 (1%) | |
329 (11%) |
305 (3%) |
291 (7%) |
319 (5%) |
321 (2%) |
323 (2%) | ||
261 (3%) |
359 (2%) |
295 (5%) |
295 (6%) |
297 (3%) |
309 (2%) | ||
241 (3%) |
289 (3%) |
281 (5%) |
281 (5%) |
281 (3%) |
297 (6%) | ||
233 (5%) |
245 (3%) |
279 (9%) |
279 (5%) |
279 (15%) |
281 (3%) | ||
207 (11%) |
125 (6%) |
263 (7%) |
267 (5%) |
261 (3%) |
279 (5%) | ||
177 (11%) |
123 (3%) |
261 (5%) |
249 (6%) |
251 (3%) |
269 (5%) | ||
123 (5%) |
121 (3%) |
169 (5%) |
195 (10%) |
195 (2%) |
263 (8%) | ||
109 (11%) |
111 (5%) |
111 (5%) |
141 (1%) |
141 (2%) |
251 (4%) | ||
97 (16%) |
97 (5%) |
97 (8%) |
127 (9%) |
123 (4%) |
243 (2%) | ||
83 (11%) |
59 (3%) |
83 (5%) |
121 (6%) |
113 (5%) |
215 (4%) | ||
59 (11%) |
59 (1%) |
101 (6%) |
101 (3%) |
213 (3%) | |||
97 (4%) |
97 (32%) |
197 (3%) | |||||
83 (2%) |
83 (2%) |
125 (4%) | |||||
59 (2%) |
59 (2%) |
111 (3%) | |||||
98 (2%) | |||||||
57 (1%) |
Standard | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
[M-H]-(%) | 335 (45%) | 319 (100%) | 319 (100%) | 429 (14%) | 415 (28%) | 445 (90%) | 445 (68%) | 429 (100%) | 445 (64%) | 397 (6%) | 385 (2%) | 397 (5%) | 451 (28%) | 287 (1%) | 431 (100%) | |
*Chain cut ions (%)
|
219 (15%) |
219 (80%) |
203 (3%) | |||||||||||||
201 (1%) |
203 (15%) |
163 (25%) | ||||||||||||||
115 (100%) |
175 (55%) |
155 (60%) | ||||||||||||||
113 (20%) |
127 (10%) | |||||||||||||||
111 (5%) | ||||||||||||||||
*Peripheral cut ions (%)
|
Loss of H
2
O
|
317 (16%) |
301 (60%) |
301 (55%) |
427 (50%) |
401 (30%) |
401 (28%) |
379 (10%) |
367 (18%) |
379 (5%) |
269 (1%) |
413 (1%) | ||||
Loss of 2H
2
O
|
299
| |||||||||||||||
Loss of CO
2
|
291 (2%) |
275 (20%) |
275 (4%) |
401 (1%) |
401 (35%) | |||||||||||
Loss of CO
2
and H
2
O
|
273 (6%) |
257 (70%) |
257 (80%) | |||||||||||||
*Loss of CO
2
and 2H
2
O
| ||||||||||||||||
*Loss of 3H
2
O
| ||||||||||||||||
Secondary daughter ions (%)
|
189 (1%) |
167 (5%) |
291 (1%) |
414 (5%) |
400 (78%) |
295 (70%) |
386 (52%) |
163 (100%) |
386 (43%) |
309 (5%) |
273 (3%) |
295 (5%) |
436 (10%) |
187 (5%) |
399 (100%) | |
163 (1%) |
149 (2%) |
171 (1%) |
163 (100%) |
175 (13%) |
149 (90%) |
179 (60%) |
135 (20%) |
179 (50%) |
213 (15%) |
259 (25%) |
253 (6%) |
241 (21%) |
173 (10%) |
393 (40%) | ||
145 (2%) |
121 (20%) |
107 (1%) |
135 (8%) |
149 (100%) |
136 (100%) |
135 (100%) |
218 (5%) |
166 (20%) |
201 (30%) |
255 (15%) |
211 (12%) |
227 (62%) |
159 (18%) |
373 (40%) | ||
99 (1%) |
99 (1%) |
59 (1%) |
121 (90%) |
121 (20%) |
107 (25%) |
123 (5%) |
135 (100%) |
173 (37%) |
213 (20%) |
159 (20%) |
223 (48%) |
145 (28%) |
355 (40%) | |||
95 (1%) |
59 (1%) |
122 (25%) |
159 (35%) |
173 (47%) |
161 (15%) |
213 (55%) |
119 (15%) |
337 (20%) | ||||||||
71 (1%) |
107 (25%) |
107 (90%) |
161 (52%) |
147 (20%) |
199 (57%) |
105 (20%) |
223 (40%) | |||||||||
59 (1%) |
81 (68%) |
159 (75%) |
107 (25%) |
187 (100%) |
95 (36%) |
85 (40%) | ||||||||||
69 (100%) |
149 (40%) |
105 (15%) |
185 (52%) |
93 (28%) | ||||||||||||
147 (72%) |
95 (38%) |
171 (55%) |
81 (58%) | |||||||||||||
107 (100%) |
93 (17%) |
71 (86%) |
69 (100%) | |||||||||||||
81 (82%) |
83 (25%) | |||||||||||||||
81 (40%) | ||||||||||||||||
36 (100%) |
Types of protons | CRC biomarker pool | Methyl esters of CRC biomarker pool |
---|---|---|
CH
3
| 0.83-0.90 | 0.83-0.90 |
CH
2
| 1.21-1.24, m | 1.21-1.24, m |
-CH
2CH2COOH | 1.57-1.65, m | 1.53-1.69, m |
-CH
2CH=CH- | 1.98-2.08, m | 1.94-2.03, m |
CH
2COO | 2.23-2.28, m | 2.23-2.31, m |
-CH=CH-CH
2-CH= | 2.75-2.79, m | 2.74-2.82, m |
OCH
3
| - | 3.64, s |
-CH(OH)CH= | 3.45-3.71, 4.03-4.26 | 4.02-4.12, 4.16-4.26, 4.58-4.60 |
-CH= | 5.10-5.47, m | 5.08-5.40, m |
-CH(OH)CH= | 5.76-5.91, m | 5.75-5.90, m |