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Multiple Stage Tandem Mass Spectrometry
for identification of plastic contaminations in blood plasma
using nano electrospray ionization
Tobias Kind and Kwang-Hyeon Liu
FiehnLab UC Davis 2009
http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/LipidAnalysis
File under: Anecdotal mass spectrometry
CC-by Free Reuse License granted; Tobias Kind FiehnLab 2009
Instrumentation – robotized chip based nano electrospray ionization
with linear iontrap
+
nanoESI chip
LTQ Linear IonTrap
Advion NanoMate robot not shown
Full scan nanoESI positive mode with data dependent
multiple stage tandem MS
Plasma_Pos_LowMS_60MSMS_CE45_01 #35-73 RT: 0.07-0.15 AV: 39 NL: 2.53E4
T: ITMS + p ESI Full ms [200.00-1200.00]
140
We will fragment this ion with the awe-inspiring power of
MS/MS/MS/MS/MS/MS/MS/MS or MS8
130
120
110
650.55
100
90
80
70
703.64
60
678.64
50
654.18
40
680.27
30
648.55
20
10
644.45
0
640
650
655.27 664.55
666.55
660.45
660
676.55
686.27 690.55
701.64
670
680
690
700
705.64
706.55
707.55
710
m/z
Slowly repeat with me: MS1..MS2..MS3..MS4..MS5..MS6..MS7..MS8...
What could it be?
Plasma_Pos_LowMS_60MSMS_CE45_01 #184 RT: 1.15 AV: 1 NL: 2.29E3
T: ITMS + c ESI d Full ms2 [email protected] [170.00-675.00]
607.32
100
-56.8
95
90
85
80
75
MS^2
precursor m/z 664.00 (ESI+)
70
Relative Abundance
65
551.24
60
55
-112.76
50
45
40
35
30
25
20
-168.74
15
495.26
10
5
0
212.44
200
359.24
283.20 325.27
250
300
350
540.52
437.36
400
450
m/z
500
550
604.28 631.35
600
650
Plasma_Pos_LowMS_60MSMS_CE45_01 #190 RT: 1.27 AV: 1 NL: 1.16E2
T: ITMS + c ESI d Full ms8 [email protected] [email protected] [email protected] [email protected] 439.26 ...
233.03
100
95
90
85
80
75
214.97
MS^8 (ESI+)
precursor m/z 327.00
70
65
60
55
50
45
40
35
30
25
153.09
20
15
251.07
291.17
171.07
10
309.12
174.99
5
0
100
150
250
200
m/z
300
Plasma_Pos_LowMS_60MSMS_CE45_01 #184-190 RT: 1.15-1.27 AV: 7 NL: 2.99E3
T: Composite spectrum MS8 664.00,607.32,551.33,495.34,439.26,383.17,327.06
551.29
100
95
90
MS^8 composite tandem mass spectrum
of precursor m/z 664.00 (ESI+)
85
80
495.31
75
607.32
70
65
60
55
50
45
40
35
327.08
30
383.17
25
439.26
56 Da
20
56 Da
56 Da
56 Da
15
56 Da
10
5
233.03
251.07
153.09
0
100
200
359.24
300
419.50
400
480.52
540.52
500
631.35
600
m/z
Sample: KH Liu (FiehnLab)
List of ions from precursor m/z 664.00 (ESI+)
Plasma_Pos_LowMS_60MSMS_CE45_01.raw
Friday, April 03, 2009
MS2 precursor 664.00
|__MS3 precursor 607.32
| |__MS4 precursor 551.33
| | |__MS5 precursor 495.34
| | | |__MS6 precursor 439.26
| | | | |__MS7 precursor 383.17
| | | | | |__MS8 precursor 327.06
| | | | | | |__Composite spectrum MS8 664.00,607.32,551.33,495.34,439.26,383.17,327.06
| | | | | | |__Single spectrum MS8 664.00,607.32,551.33,495.34,439.26,383.17,327.06 (190)
| | | | | |__Composite spectrum MS7 664.00,607.32,551.33,495.34,439.26,383.17
| | | | | |__Single spectrum MS7 664.00,607.32,551.33,495.34,439.26,383.17 (189)
| | | | |__Composite spectrum MS6 664.00,607.32,551.33,495.34,439.26
| | | | |__Single spectrum MS6 664.00,607.32,551.33,495.34,439.26 (188)
| | | |__Composite spectrum MS5 664.00,607.32,551.33,495.34
| | | |__Single spectrum MS5 664.00,607.32,551.33,495.34 (187)
| | |__Composite spectrum MS4 664.00,607.32,551.33
| | |__Single spectrum MS4 664.00,607.32,551.33 (186)
| |__Composite spectrum MS3 664.00,607.32
| |__Single spectrum MS3 664.00,607.32 (185)
|__Single spectrum MS2 664.00 (184)
List of product ions:
m/z 664 , 607 ,551 ,495 ,439 ,383 ,327
accurate masses see above
Solutions – well Plasma – well Lipids use LipidMaps tools!
CE(18:2(Ep))
CE(18:2(Ke))
CE(18:3(OH))
CE(18:1(Ep))-cyclo
CE(18:1(Ke))-cyclo
CE(18:2(OH))-cyclo
CE(16:2(Ke,Ep2))
CE(16:2(Ke2,Ep))
CE(16:3(Ep,OO))
CE(16:3(Ke,OO))
CE(16:3(OH,Ep2))
CE(16:3(OH,Ke,Ep))
CE(16:3(OH,Ke2))
CE(16:4(OOH,Ep))
CE(16:4(OOH,Ke))
CE(16:4(OH,OO))
CE(16:4(OH2,Ep))
CE(16:4(OH2,Ke))
CE(16:5(OH,OOH))
CE(16:5(OH3))
CE(16:1(Ke,Ep2))-cyclo
CE(16:1(Ke2,Ep))-cyclo
CE(16:2(Ep,OO))-cyclo
CE(16:2(Ke,OO))-cyclo
CE(16:2(OH,Ep2))-cyclo
CE(16:2(OH,Ke,Ep))-cyclo
CE(16:2(OH,Ke2))-cyclo
CE(16:3(OOH,Ep))-cyclo
CE(16:3(OOH,Ke))-cyclo
CE(16:3(OH,OO))-cyclo
CE(16:3(OH2,Ep))-cyclo
CE(16:3(OH2,Ke))-cyclo
CE(16:4(OH,OOH))-cyclo
CE(16:4(OH3))-cyclo
18
18
18
18
18
18
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
2
2
3
1
1
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
1
1
2
2
2
2
2
3
3
3
3
3
4
4
663.5711
663.5711
663.5711
663.5711
663.5711
663.5711
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
663.4983
0.4289
0.4289
0.4289
0.4289
0.4289
0.4289
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
0.5017
C45H75O3+
C45H75O3+
C45H75O3+
C45H75O3+
C45H75O3+
C45H75O3+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
C43H67O5+
Current mass accuracy for unit mass resolution LTQ:
78 ppm (external calibration) or 0.046 Da @ 594.46 Da for reference purposes
LipidMaps Tools used with M+H
Solutions – well Plasma – well Lipids!
CE(18:2(Ep))
CE(18:2(Ke))
CE(18:3(OH))
CE(18:1(Ep))-cyclo
CE(18:1(Ke))-cyclo
CE(18:2(OH))-cyclo
18
18
18
18
18
18
2
2
3
1
1
2
663.5711
663.5711
663.5711
663.5711
663.5711
663.5711
0.4289
0.4289
0.4289
0.4289
0.4289
0.4289
C45H75O3+
C45H75O3+
C45H75O3+
C45H75O3+
C45H75O3+
C45H75O3+
Sphingosine
Sphingosine
Sphinganine
CerP
HexCer
Cer
18:1
12:0
24:4
644.5013
644.5096
644.5976
0.5013
0.5096
0.5976
C36H71NO6P+
C36H70NO8+
C42H78NO3+
Problem: Our mass accuracy is 0.046 Da but the solutions are 0.5 Da difference
Without Google Scholar we would be lost to search single m/z values
or numbers. Still its a horrible way to do mass spectral library search.
Has anybody a Google API example?
Solutions – well Plasma – well Google It!
On the lipid composition of human meibum and tears:
Comparative analysis of nonpolar lipids
Igor A. Butovich
http://www.iovs.org/cgi/content/abstract/iovs.08-1889v1
"However, rushing to this conclusion would have been a mistake because subsequent fragmentation
of ion m/z 663 in sequential MSn experiments gave us the following chain of product ions
differing by 56 amu: 663 (MS1) -> 607 (MS2) -> 551 (MS3) -> 495 (MS4) -> 439 (MS5) -> 383 (MS6) -> 327 (MS7)
(not shown). Note that the first four ions (663, 607, 551, 495) were also easily detectable in a simple MS1 experiment
(Fig. 11A) . These transformations were indicative of a sequential loss of several up to six) t-butyl groups and
were formerly described for oxidized form of Irgafos 168 (C42H63O4P, Fig. 11A , inset) a
polymer additive present in polyethylene and polypropylene and its derivatives."
Researcher working with tears = The Dacriologist (Book by Djerassi)
Dakryology, the neologism for the science of tears
10.1007/BF00137944
also CRYING: The Natural and Cultural History of Tears By Tom Lutz Norton
Solutions – well Plasma – well Plasticizers!
Analysis of Additives in Polyethylene with Desorption
Chemical Ionization/Tandem Mass Spectrometry
S. W. CHEN and G. R. HER*
Appl. Spectrosc. 47, 844-851 (1993)
http://www.opticsinfobase.org/abstract.cfm?URI=as-47-6-844
Picture Source: S. W. CHEN and G. R. HER Appl. Spectrosc. 47, 844-851 (1993)
Also Oxidized Naugard 524 - http://dx.doi.org/10.1016/0003-2670(95)00183-Z
Solution – probably Irganox / Irgafos contamination
551
Plasma_Pos_LowMS_60MSMS_CE45_01 #184-190 RT: 1.15-1.27 AV: 7 NL: 2.99E3
T: Composite spectrum MS8 664.00,607.32,551.33,495.34,439.26,383.17,327.06
100
70
60
30
439
40
383
50
327
Relative Abundance
80
607
495
90
Our MS^8
"Lipid"
20
10
0
100
200
300
400
500
600
m/z
Picture Source: Analysis of Additives in Polyethylene with Desorption
Chemical Ionization/Tandem Mass Spectrometry
S. W. CHEN and G. R. HER*
Appl. Spectrosc. 47, 844-851 (1993)
http://www.opticsinfobase.org/abstract.cfm?URI=as-47-6-844
Problem solved, "lipid" is oxidized plasticizer.
Is the problem really solved?
No, because for the formula = C42H63O4P
the exact mass = 662.446397016.
The [M+H]+ would be 663.4542 Da
and not 664.0
CID 14572930
In this case an ultrazoom investigation or
infusion into FT-ICR-MS with resolving power
50,000 to 100,000 would probably help.
InChIKey: AZSKHRTUXHLAHS-UHFFFAOYSA-N
MS/MS on isotopic peak, ion suppression of isobars, overlap with
multiple ceramides or cholesterol and oxycholesterol species possible
Final question – How can we avoid rediscovery (reinventing the wheel)?
• sharing experiments (free)
• sharing mass spectra (free or sell)
• putting publications into OA for easier searching
How can we prevent plasticizer contaminations?
• running solvent blanks with each experiment
• running methods blanks with each experiment
• using only (baked) glassware
Other solutions?
• library search (but compound is not in NIST)
• remember the awesome power of MS/MS/MS/MS/MS/MS/MS/MS
• modern iontraps are currently not used to the full extend Data Dependent
• exclusion libraries for unwanted "contaminations"
See: XLS supplement from Keller, B.O.; Sui, J.; Young, A.B.; Whittal, R.M.
Interferences and contaminants encountered in modern mass spectrometry.