Transcript Document

Biology
Chemistry
Informatics
Welcome!
Mass Spectrometry meets Cheminformatics
WCMC Metabolomics Course 2013
Tobias Kind
Course 6: Concepts for LC-MS
http://fiehnlab.ucdavis.edu/staff/kind
1
CC-BY License
General LC-MS data processing for small molecules
Confirm with MS/MS
or MSn fragmentation
100
50
MS2
0
200
400
600
2
Deconvolution and evaluation of LC-MS data
UPLC_C8_DataDependent_Chlamy_07030703...
3/7/2007 3:21:28 AM
RT: 0.00 - 44.99
100
17.80
90
NL: 1.68E6
18.30
m/z= 70.00000-2000.00000
F: FTMS + p NSI Full ms
[200.00-1200.00] MS
UPLC_C8_DataDependent
_Chlamy_070307032128
19.11
LC-MS
Chromatogram
80
70
Picture: nrel.gov
60
29.36
LC-MS run 40 minutes
C8 column, Agilent-UPLC
Chlamydomonas extract
28.74
50
20.16
40
30.30
11.27
43.97
30.55
23.00
30
17.49
43.68
23.80
20
10.31
10
1.56
6.37
16.07
9.55
31.53
43.09
37.19
0
0
5
10
15
20
25
Time (min)
30
35
40
+
UPLC_C8_DataDependent_Chlamy_070307032128 #1414 RT: 18.30 AV: 1 NL: 8.23E5
F: FTMS + p NSI Full ms [200.00-1200.00]
756.57745
R=55401
100
90
80
Extracted
Mass spectrum
70
60
757.58209
R=55100
50
40
30
20
756.26086
R=87000
10
754.57471
R=50200
0
754
755
755.57501
R=60200
756
758.58636
R=56100
756.89417
R=86600
757
759.59546
R=60400
757.90027
R=79200
758
759
m/z
Chromatogram Source: N.Saad, DY Lee FiehnLab
760
760.60144 761.59888 762.29700
R=52200 R=62600 R=49901
761
762
FT-ICR-MS mass spectrum
MS1 @ 50,000 resolving power
Check charge state = 1
756.57 represents [M+H]+
3
Peak Picking with ACD/SpecManager 9.0
4
Processing of LC-MS data - use of MassFrontier
5
Deconvolution and evaluation of LC-MS data
Example with HighChem Mass Frontier
LC-MS detected compound
Marked with blue triangle
LC-MS detected compound
Marked with blue triangle
141 peaks extracted
Extracted MS1 peak
Library search useless
(only single peak)
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Adduct removal and detection during ESI-LC-MS runs
100
756.57745
R=55401
[M+H]+ = 756.577
M = 755.5627
90
80
Relative Abundance
70
60
757.58209
R=55100
50
40
30
20
758.58636
R=56100
10
759.59546
R=60400
0
756.5
Ion name
757.0
757.5
Ion mass
758.0
m/z
758.5
759.0
759.5
Your M here:
755.562724
Your M+X or M-X
756.577
Charge
Mult Mass
Result:
Reverse:
1. Positive ion mode
M+2H
M/2 + 1.007276
M+H+NH4
M/2 + 9.520550
M+H+Na
M/2 + 11.998247
M+H
M + 1.007276
M+NH4
M + 18.033823
M+Na
M + 22.989218
2+
2+
2+
1+
1+
1+
0.5
0.5
0.5
1
1
1
1.007276
9.520550
11.998247
1.007276
18.033823
22.989218
378.788638
387.301912
389.779609
756.570000
773.596547
778.551942
377.277724
368.764450
366.286753
755.562724
738.536177
733.580782
2. Negative ion mode
M-3H
M/3 - 1.007276
M-2H
M/2 - 1.007276
M-H2O-H
M- 19.01839
M-H
M - 1.007276
M+Na-2H
M + 20.974666
M+Cl
M + 34.969402
321111-
0.33
0.5
1
1
1
1
-1.007276
-1.007276
-19.01839
-1.007276
20.974666
34.969402
250.846965
376.774086
736.544334
754.555448
776.537390
790.532126
253.197276
379.292276
775.588390
757.577276
735.595334
721.600598
Download Adduct-Calculator [LINK]
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Problem: Detection of molecular ion
Problem: Is this the pure mass spectrum or from overlapping peaks?
Is it M+H or M+Na or any of the 40 other adducts?
Example data from crocin standard mixture (expected MW: 976.965)
27-2-crocin-LTQFT-pos-0V #1 RT: 0.00 AV: 1
T: FTMS + p NSI Full ms [ 50.00-2000.00]
27-2-crocin-LTQFT-pos-100V #1 RT: 0.01 AV: 1 NL: 1.30E4
T: FTMS + p NSI sid=100.00 Full ms [ 50.00-1500.00]
NL: 9.78E3
719.21790
100
[M+Na]+
95
95
90
999.37189
100
90
CID = 0V
85
85
80
80
75
75
70
70
65
65
60
60
CID = 100V
Relative Abundance
719.21747
55
50
45
55
749.22906
50
45
529.17432
40
40
1035.36377
367.12146
575.21149
35
35
30
25
30
[M+Na]+
853.28149
1015.33575
675.26556
529.17535
25
853.28583
20
20
367.12228
15
575.21246
1177.38086
301.14145
1241.42676
10
10
1359.45837
1517.50293
5
0
400
600
800
1000
m/z
1200
1400
1600
329.95432
219.18610
5
1695.55408
200
1177.39673
15
185.11496
1800
0
2000
969.36615
200
8
485.20132
77.14542
400
600
800
m/z
1000
1225.41028
1339.45142
1200
1400
Adduct removal and detection during LC-MS runs
Ion name
Ion mass
1. Positive ion mode
M+2H
M/2 + 1.007276
M+H+NH4
M/2 + 9.520550
M+H+Na
M/2 + 11.998247
M+H
M + 1.007276
M+NH4
M + 18.033823
M+Na
M + 22.989218
2. Negative ion mode
M-3H
M/3 - 1.007276
M-2H
M/2 - 1.007276
M-H2O-H
M- 19.01839
M-H
M - 1.007276
M+Na-2H
M + 20.974666
M+Cl
M + 34.969402
Charge
Mult Mass
Your M here:
853.33089
Your M+X or M-X
756.577
Result:
Reverse:
2+
2+
2+
1+
1+
1+
0.5
0.5
0.5
1
1
1
1.007276
9.520550
11.998247
1.007276
18.033823
22.989218
427.672721
436.185995
438.663692
854.338166
871.364713
876.320108
437.152724
428.639450
426.161753
875.312724
858.286177
853.330782
321111-
0.33
0.5
1
1
1
1
-1.007276
-1.007276
-19.01839
-1.007276
20.974666
34.969402
251.182724
377.277724
737.551610
755.562724
777.544666
791.539402
253.197276
379.292276
775.588390
757.577276
735.595334
721.600598
Adduct can be removed before or after formula generation.
For good isotopic pattern matching remove adduct after formula generation.
7 Golden Rules apply LEWIS and SENIOR check (adduct needs to be removed)
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Formula Generation from accurate mass measurement
CHN5O13P15 or CHN11P19 ???
Apply Seven Golden Rules for correct molecular formulas
Apply heuristic and mathematical and chemometric rules for filtering elemental compositions
10
Isotopic Pattern generator from formula
Example from MWTWIN
Is usually included in every LC-MS software
11
Isotopic pattern equally important as accurate mass
Experimental result
Abundances for all molecular formulae
756.57745
R=55401
100
90
80
A+1 = 47.44%
Relative Abundance
70
60
757.58209
R=55100
50
40
A+2 = 10.92 %
30
20
758.58636
R=56100
10
759.59546
R=60400
0
756.5
757.0
757.5
758.0
m/z
m/z
Intensity
Relative
Resolution
756.5765
757.5814
758.5862
756.5526
759.593
589034.1
279455.2
64293.3
19173.8
9656.4
100
47.44
10.92
3.26
1.64
54901
55100
56100
52000
56000
758.5
759.0
759.5
We can discard all other results
outside the error box.
Current box reflect +/- 10% error.
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Problems during LC-MS peak detection and MS deconvolution
48
Multiple peaks detected
But it is a single component only
36
24
12
0
10.80
10.90
11.00
11.10
11.20
11.30
11.40
11.50
11.60
11.70
11.80
11.90
Multiple peaks detected – Solution: adjust deconvolution settings
Mass spectra not clean – Solution: manual peak extraction
Not enough peaks detected – Solution: increase signal noise (S/N) settings
Finding optimum settings is:
• non-trivial and can change in different matrices
• can be evaluated on standards and quality check mixtures
• can be obtained by self-sharpening algorithms
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UPLC-FT-MS data extraction with MassFrontier
Mass Frontier 5.0 Report
File MS1 FTMS + p NSI Full #1414
MS1
100
Fragment peaks m/z 478.45 and 496.46
756.577
50
0
755
100
760
478.45
496.46
75
MS2
File MS 2 ITMS + c NSI d Full 756.58 #1415
100
MS2
50
478.45 496.46
50
25
335.56
0
200
400
600
0
236.20
271.06
250
300
335.56
350
391.34 434.52
400
450
500
550
600
650
687.72
700
100
756.577
75
MS1
50
25
0
751
752
753
754
755
756
757
758
759
760
761
762
Approach: generate molecular formula using Seven Golden Rules;
find matching isomers in molecular databases;
confirm possible matches by in-silico fragmentation (usually impossible);
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Seven Golden Rules – generate possible molecular formulas
5 formula candidates left with 30 ppm mass accuracy and 10% isotopic abundances
These are candidates with good isotopic pattern match. These 5 were found in PubChem.
C42H78NO8P - 1 isomer hit
C42H77NO10 - 1 isomer hit
C39H73N5O9 - 0 isomer hit
C43H82NO7P - 2 isomers found
C43H73N5O6 -2 isomers found
C45H77N3O6 - 1 hit found
C45H69N7O3 - 1 hit found
Scan speed problem:
Due to poor ion statistics only few scans are collected
Mass accuracy and isotopic abundance accuracy are bad
15
Structural isomer lookup example in ChemSpider
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In-silico fragmentation with MassFrontier
using fragmentation library of 20,000 mechanisms from literature
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In-silico fragmentation with MassFrontier
Experimental peaks m/z 478.45 and 496.46 were detected in MS/MS spectrum
In-silico fragmentation should match the experimental fragmentation.
In-silico - using a computer library of 20,000 fragmentation rules from the MS literature
C42H78NO8P – Fragments N.A.
C42H77NO10 – Fragments N.A.
C43H82NO7P – Fragments m/z 478 and 496
LWHKEEJOPDYARM-WYCAKVPNBV
Possible solution (2 fragments match)
C45H69N7O3 – Fragments: m/z 496
C43H82NO7PH – Fragments N.A.
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Discussion of general LC-MS approach
Example discussion:
Fragment at m/z 236 not explained; molecular ion may be wrong;
Substance can be potential new compound or not in database
Must be confirmed by NMR or external standard
General problems:
Best approach is to generate MS and MSn and MSe mass spectral libraries
Adduct removal is a problem
Building target lists is always good (know what to expect)
Focus on certain substance classes only
Focus on single compound only
Substance must be known for in-silico approach
Fragmentation rules must be captured for in-silico approach
In-silico approaches work best for peptides, carbohydrates, lipids
(due to known and stable fragmentations)
Importance:
Taxonomics species-compounds relationship databases or pathway DBs
KNApSAcK/ database, KEGG, MetaCyc
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All theory is lost – if compound is truly unknown or
not in public database
92.10
92.07
92.05
91.99
91.92
91.90
91.87
NO
NO
NO
NO
NO
NO
NO
C44H70N9P
C43H84NOP3S
C37H93NO3P4S
C46H77NO7
C33H80N11P3S
C36H82N7O5PS
C37H86N5P5
Rank 147 in 7GR with original settings
NO
NO
NO
NO
NO
NO
NO
C46H77NO7 not found in ChemSpider, PubChem, LipidMaps (2013)
Only found in internal LipidBlast database.
UPLC-FTICR settings readjusted to
3 ppm mass accuracy and
5% isotopic abundance error
Actual mass error is: -1.718266 ppm
Max. isotopic abundance error: 4.10 0%
Additional evidence, there is not PC in
Chlamydomonas, but mostly the betaine lipid DGTS
Rank 18 in 7GR with original settings (from 56)
DGTS accounts for about 10% of total lipid in Chlamydomonas; Peter Schlapfer, Waldemar Eichenberger
Plant Science Letters Volume 32, Issues 1-2, October-November 1983, Pages 243-252 and
FEBS Letters, Volume 88, Issue 2, 15 April 1978, Pages 201–204; W. Eichenberger , A. Boschetti
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LipidBlast comes to help unravel the mystery
best tentative structure proposal so far
Experimental MS/MS
m/z 236 instead of m/z 184
DGTS instead of PC
Precursor m/z
756.576500 (M+H)+
LipidBlast in-silico MS/MS
Name: DGTS 36:6; [M+H]+; DGTS(18:3/18:3)
MW: 756 Exact Mass: 756.57782 ID#: 9031 DB: dgts+hpos-it.msp
Comment: Parent=756.57782 Mz_exact=756.57782 ;
DGTS 36:6; [M+H]+; DGTS(18:3/18:3); C46H77NO7
7 m/z Values and Intensities:
236.14979
200.00
478.35339
600.00
496.36395
600.00
625.48320
400.00
639.49885
250.00
738.56726
999.00
756.57782
10.00
C10H21NO5H+ (236.14)
[M+H]-sn1-H2O || [M+H]-sn2-H2O
[M+H]-sn1 || [M+H]-sn2
[M+H]-131
[M+H]-117
[M-H2O]+
[M+H]
Betaine lipid DGTS(18:3/18:3)
Kind T, Liu KH, Lee do Y, Defelice B, Meissen JK, Fiehn O.
Nature Methods. 2013 Aug;10(8):755-8. doi: 10.1038/nmeth.2551. Epub 2013 Jun 30.
LipidBlast in silico tandem mass spectrometry database for lipid identification.
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The Last Page - What is important to remember:
Always use peak picking and mass spectral deconvolution for LC-MS data
Apply accurate mass, accurate isotopic abundances together for formula generation
Make use of high resolving power whenever possible
Use MS/MS data and mass spectra from different ionization voltages
Use existing MS/MS libraries or create your own MSn tree libraries
Use molecular isomer databases for obtaining possible structure candidates
Confirm if possible with MSn data or other possible filter constraints
Validation, validation, validation: Pipelines and workflows must be validated with
unknown (unknown) compounds
Compound is tentative until finally approved with reference standard or matching
with multiple orthogonal parameters such as MS/MS, retention time or NMR.
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Recent literature (2012/2013)
Applying Tandem Mass Spectral Libraries for Solving the Critical Assessment of Small Molecule Identification (CASMI) LC/MS
Challenge 2012; Oberacher H; Metabolites 2013, 3(2), 312-324; doi:10.3390/metabo3020312
Computational mass spectrometry for small molecules; J Cheminform. 2013; 5: 12.; Kerstin Scheubert, Franziska Hufsky, Sebastian Böcker
Published online 2013 March 1. doi: 10.1186/1758-2946-5-12
A Rough Guide to Metabolite Identification Using High Resolution Liquid Chromatography Mass Spectrometry in Metabolomic
Profiling in Metazoans; Computational and Structural Biotechnology Journal ;David Watson; Volume No: 4, Issue: 5, January 2013, e201301005,
http://dx.doi.org/10.5936/csbj.201301005
Bioinformatics. 2011 Apr 15;27(8):1108-12. doi: 10.1093/bioinformatics/btr079. Epub 2011 Feb 16.
Brown M, Wedge DC, Goodacre R, Kell DB, Baker PN, Kenny LC, Mamas MA, Neyses L, Dunn WB.
Automated workflows for accurate mass-based putative metabolite identification in LC/MS-derived metabolomic datasets.
{PUTMEDID_LCMS}
IDEOM: an Excel interface for analysis of LC–MS-based metabolomics data;
Bioinformatics. 2012 Apr 1;28(7):1048-9. doi: 10.1093/bioinformatics/bts069. Epub 2012 Feb 4.
IDEOM: an Excel interface for analysis of LC-MS-based metabolomics data.
Creek DJ, Jankevics A, Burgess KE, Breitling R, Barrett MP.
Anal Chem. 2012 Jan 3;84(1):283-9. doi: 10.1021/ac202450g. Epub 2011 Dec 12.
CAMERA: an integrated strategy for compound spectra extraction and annotation of liquid chromatography/mass spectrometry data
sets. Kuhl C, Tautenhahn R, Böttcher C, Larson TR, Neumann S.
Mass appeal: metabolite identification in mass spectrometry-focused untargeted metabolomics;
March 2013, Volume 9, Issue 1 Supplement, pp 44-66; Warwick B. Dunn, Alexander Erban, Ralf J. M. Weber, Darren J. Creek, Marie Brown,
Rainer Breitling, Thomas Hankemeier, Royston Goodacre, Steffen Neumann, Joachim Kopka, Mark R. Viant
Metabolite profiling and beyond: approaches for the rapid processing and annotation of human blood serum mass spectrometry data;
Metabolomics and Metabolite Profiling Analytical and Bioanalytical Chemistry
June 2013, Volume 405, Issue 15, pp 5037-5048 [LINK]
Anal Chem. 2013 Apr 2;85(7):3576-83. doi: 10.1021/ac303218u. Epub 2013 Mar 21.
Automated pipeline for de novo metabolite identification using mass-spectrometry-based metabolomics.
Peironcely JE, Rojas-Chertó M, Tas A, Vreeken R, Reijmers T, Coulier L, Hankemeier T.
http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/Structure_Elucidation/
23