Biomedical Engineering Reference
In-Depth Information
Ion chromatogram
46.75
1.19e6
1.00e6
1.21
9.34
8.00e5
45.05
13.72
6.00e5
24.04
48.91
22.10
15.53
17.15
11.65
17.95
30.41
37.12
4.00e5
4.78
31.86
29.26
0
.
40
27.47
2.00e5
0.00
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
Time, min
MS full scan at 17.2 minutes
461.75(2
+
)
400
300
200
MS
(selection of MRM
precursor ion)
400
380
360
340
320
300
280
260
240
220
200
180
160
140
120
10
8
60
4
2
0
461.7515(2)
722.41(1
+
)
100
0
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
462.2518(2)
m/z, Da
MSMS full scan of 416.1
CID
201.10
132
722.39
477.2108
462.7539(2)
slelection for MRM
fragment ion
slelection for MRM
fragment ion
100
454
456
458
460
462
464
466
468
470
472
474
476
478
480
m/z, Da
120.09
155.09
50
476.27
575.33
376.19
248. 17
0
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
m/z, Da
Design MRM
2.0e6
1.9e6
1.8e6
1.7e6
1.6e6
1.5e6
1.4e6
1.3e6
1.2e6
1.1e6
1.0e6
9.0e6
8.0e6
7.0e6
6.0e6
5.0e6
4.0e6
3.0e6
2.0e6
1.0e6
0.0
14.82
461/722 (precursor/fragment ion)
MRM with selected
precursor ion and two fragment ions
461/575 (precursor/fragment ion)
2.5e6
2.0e6
1.5e6
1.0e6
5.0e5
0.0
14.82
408
14.3
14.4
14.5
14.6
14.7
14.8
14.9
15.0
15.1
15.2
15.3
15.4
410
413
416
419
422
424
427
430
433
436
439
Time, min
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
58
116
173
231
287
344
399
455
512
568
622
678
734
792
849
907
964
Time, min
Fig. 10.2 Selection and design of transitions from a LC-MS/MS experiment. Peptides from discovery-based experiments
are identified based on their fragmentation pattern. When a specific peptide is eluting from the reverse-phase column the
resulting ion current is detected (in the ion chromatogram). Subsequently, the double-charged peptide is selected (m/z
461.75) for fragmentation by collision-induced fragmentation (CID). The resulting fragmentation pattern is subsequently
used for peptide identification. Transitions are selected from the precursor ion mass to charge (m/z 461.75) and the
resulting fragment ions (m/z 722.39 and 575.33). When the same sample is analyzed by an MRM assay, specific for
these particular transitions, all other ions are ignored. As can be seen from the insert, the transitions co-elute. In addition,
the relative abundance of the signals from the transitions corresponds to the relative abundance of the fragment ions in
the MS/MS spectrum.
establishing an assay using data generated from the biological sys-
tem of interest. Prior analysis ensures that the sample preparation
protocol is suitable for mass spectrometric analysis and that the
subset of proteins and peptides selected for monitoring by MRM
can be detected by the mass spectrometer.
In addition, by using existing LC-MS/MS data, potential
interference from isobaric peptides, splice variants, etc., can bet-
ter be taken into account. However, the potential for interference
from these peptides depends greatly on the sample type that is
analyzed. For example, the complexity of a digested cell lysate can
mask the presence of isobaric peptides and splice variants, whereas
analysis of a purified protein is more likely to reveal if interfering
peptides are present.
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