mass spectrometry meets cheminformatics tobias kind...
Post on 29-Mar-2018
214 Views
Preview:
TRANSCRIPT
Welcome!
Mass Spectrometry meets CheminformaticsTobias Kind and Julie Leary
UC Davis
Course 7: Concepts for LC-MS
Class website: CHE 241 - Spring 2008 - CRN 16583Slides: http://fiehnlab.ucdavis.edu/staff/kind/Teaching/PPT is hyperlinked – please change to Slide Show Mode
General LC-MS data processing for small molecules
Confirm with MS/MS or MSn fragmentation
100
0
50
200 400 600
MS2
UPLC_C8_DataDependent_Chlamy_07030703... 3/7/2007 3:21:28 AM
RT: 0.00 - 44.99
0 5 10 15 20 25 30 35 40Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
18.3017.80
19.11
29.36
28.7420.16 30.30
11.27 43.9730.5523.00
43.6817.49 23.80
10.31 16.079.55 31.531.56 6.37 43.0937.19
NL: 1.68E6m/z= 70.00000-2000.00000 F: FTMS + p NSI Full ms [200.00-1200.00] MS UPLC_C8_DataDependent_Chlamy_070307032128
UPLC_C8_DataDependent_Chlamy_070307032128 #1414 RT: 18.30 AV: 1 NL: 8.23E5F: FTMS + p NSI Full ms [200.00-1200.00]
754 755 756 757 758 759 760 761 762m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
756.57745R=55401
757.58209R=55100
758.58636R=56100
759.59546R=60400
762.29700R=49901
755.57501R=60200
756.89417R=86600
760.60144R=52200
756.26086R=87000
754.57471R=50200
757.90027R=79200
761.59888R=62600
LC-MS run 40 minutesC8 column, Agilent-UPLCChlamydomonas extract
FT-ICR-MS mass spectrumMS1 @ 50,000 resolving powerCheck charge state = 1756.57 represents [M+H]+
+
Deconvolution and evaluation of LC-MS data
Picture: nrel.gov
Chromatogram Source: N.Saad, DY Lee FiehnLab
LC-MSChromatogram
ExtractedMass spectrum
Peak Picking with ACD/SpecManager 9.0
Processing of LC-MS data - use of MassFrontier
100
0
50
75
25
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
100
0
50
75
25
200 300 400 500 600 700 800 900 1000 1100 1200
756.577
706.568585.457 871.588324.209 436.437 951.566 1107.90237.733 1196.27516.755 805.909
Deconvolution and evaluation of LC-MS data
LC-MS detected compoundMarked with blue triangle
Extracted MS1 peakLibrary search useless(only single peak)
LC-MS detected compoundMarked with blue triangle141 peaks extracted
Example with HighChem Mass Frontier
Adduct removal and detection during ESI-LC-MS runs
756.5 757.0 757.5 758.0 758.5 759.0 759.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
756.57745R=55401
757.58209R=55100
758.58636R=56100
759.59546R=60400
756.5 757.0 757.5 758.0 758.5 759.0 759.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
756.57745R=55401
757.58209R=55100
758.58636R=56100
759.59546R=60400
[M+H]+ = 756.577
Download Adduct-Calculator [LINK]
M = 755.5627
Your M here: Your M+X or M-X755.562724 756.577
Ion name Ion mass Charge Mult Mass Result: Reverse:
1. Positive ion mode M+2H M/2 + 1.007276 2+ 0.5 1.007276 378.788638 377.277724M+H+NH4 M/2 + 9.520550 2+ 0.5 9.520550 387.301912 368.764450M+H+Na M/2 + 11.998247 2+ 0.5 11.998247 389.779609 366.286753M+H M + 1.007276 1+ 1 1.007276 756.570000 755.562724M+NH4 M + 18.033823 1+ 1 18.033823 773.596547 738.536177M+Na M + 22.989218 1+ 1 22.989218 778.551942 733.580782
2. Negative ion mode M-3H M/3 - 1.007276 3- 0.33 -1.007276 250.846965 253.197276M-2H M/2 - 1.007276 2- 0.5 -1.007276 376.774086 379.292276M-H2O-H M- 19.01839 1- 1 -19.01839 736.544334 775.588390M-H M - 1.007276 1- 1 -1.007276 754.555448 757.577276M+Na-2H M + 20.974666 1- 1 20.974666 776.537390 735.595334M+Cl M + 34.969402 1- 1 34.969402 790.532126 721.600598
Problem: Detection of molecular ion
2 7 - 2 -c ro c in -L T Q F T - p o s -0 V # 1 R T : 0 .0 0 A V : 1 N L : 9 .7 8 E 3T : F T M S + p N S I F u l l m s [ 5 0 .0 0 -2 0 0 0 .0 0 ]
2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0m / z
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
1 0 0
Re
lativ
e A
bu
nd
an
ce
7 1 9 .2 1 7 9 0
5 2 9 . 1 7 4 3 2
3 6 7 . 1 2 1 4 65 7 5 . 2 1 1 4 9
1 0 1 5 . 3 3 5 7 58 5 3 .2 8 1 4 9
1 8 5 .1 1 4 9 6
1 1 7 7 . 3 8 0 8 63 0 1 .1 4 1 4 5
1 3 5 9 .4 5 8 3 71 5 1 7 .5 0 2 9 3
1 2 4 1 . 4 2 6 7 6
1 6 9 5 . 5
27-2-crocin-LTQFT-pos-100V #1 RT: 0.01 AV: 1 NL: 1.30E4T: FTMS + p NSI sid=100.00 Full ms [ 50.00-1500.00]
200 400 600 800 1000 1200 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
999.37189
719.21747
749.22906
853.28583529.17535675.26556
367.12228 1177.39673
575.21246
485.20132219.18610
1035.36377
969.36615
329.95432
1339.451421225.4102877.14542
[M+Na]+
[M+Na]+
CID = 0V CID = 100V
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)
Adduct removal and detection during LC-MS runsYour M here: Your M+X or M-X
853.33089 756.577
Ion name Ion mass Charge Mult Mass Result: Reverse:1. Positive ion mode M+2H M/2 + 1.007276 2+ 0.5 1.007276 427.672721 437.152724M+H+NH4 M/2 + 9.520550 2+ 0.5 9.520550 436.185995 428.639450M+H+Na M/2 + 11.998247 2+ 0.5 11.998247 438.663692 426.161753M+H M + 1.007276 1+ 1 1.007276 854.338166 875.312724M+NH4 M + 18.033823 1+ 1 18.033823 871.364713 858.286177M+Na M + 22.989218 1+ 1 22.989218 876.320108 853.330782
2. Negative ion mode M-3H M/3 - 1.007276 3- 0.33 -1.007276 251.182724 253.197276M-2H M/2 - 1.007276 2- 0.5 -1.007276 377.277724 379.292276M-H2O-H M- 19.01839 1- 1 -19.01839 737.551610 775.588390M-H M - 1.007276 1- 1 -1.007276 755.562724 757.577276M+Na-2H M + 20.974666 1- 1 20.974666 777.544666 735.595334M+Cl M + 34.969402 1- 1 34.969402 791.539402 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)
Formula Generation from accurate mass measurement
CHN5O13P15 or CHN11P19 ???
Apply Seven Golden Rules for correct molecular formulasApply heuristic and mathematical and chemometric rules for filtering elemental compositions
Isotopic Pattern generator from formula
Example from MWTWINIs usually included in every LC-MS software
Isotopic pattern equally important as accurate mass
0.00
5.00
10.00
15.00
20.00
25.00
0.00 20.00 40.00 60.00 80.00
Isotopic Abundance A+1 [%]
Isot
opic
Abu
ndan
ce A
+2 [%
]
756.5 757.0 757.5 758.0 758.5 759.0 759.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
756.57745R=55401
757.58209R=55100
758.58636R=56100
759.59546R=60400
756.5 757.0 757.5 758.0 758.5 759.0 759.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
756.57745R=55401
757.58209R=55100
758.58636R=56100
759.59546R=60400
m/z Intensity Relative Resolution
756.5765 589034.1 100 54901757.5814 279455.2 47.44 55100758.5862 64293.3 10.92 56100756.5526 19173.8 3.26 52000759.593 9656.4 1.64 56000
A+1 = 47.44%
A+2 = 10.92 %
We can discard all other resultsoutside the error box.Current box reflect +/- 10% error.
Experimental result Abundances for all molecular formulae
48
0
24
36
12
10.80 10.90 11.00 11.10 11.20 11.30 11.40 11.50 11.60 11.70 11.80 11.90
Problems during LC-MS peak detection and MS deconvolution
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
Multiple peaks detectedBut it is a single component only
UPLC-FT-MS data extraction with MassFrontier
100
0
50
75
25
250 300 350 400 450 500 550 600 650 700
478.45496.46
236.20
434.52391.34 687.72335.56271.06
Mass Frontier 5.0 Report
File MS1 FTMS + p NSI Full #1414
100
0
50
755 760
756.577
File MS 2 ITMS + c NSI d Full 756.58 #1415
100
0
50
200 400 600
478.45 496.46
335.56
100
0
50
75
25
751 752 753 754 755 756 757 758 759 760 761 762
756.577MS1
MS2
MS1 Fragment peaks m/z 478.45 and 496.46
MS2
Approach: generate molecular formula using Seven Golden Rules;find matching isomers in molecular databases;confirm possible matches by in-silico fragmentation (usually impossible);
Seven Golden Rules – generate possible molecular formulas
5 formula candidates left with 30 ppm mass accuracy and 10% isotopic abundancesThese are candidates with good isotopic pattern match. These 5 were found in PubChem.C42H78NO8P - 1 isomer hitC42H77NO10 - 1 isomer hitC39H73N5O9 - 0 isomer hitC43H82NO7P - 2 isomers foundC43H73N5O6 -2 isomers foundC45H77N3O6 - 1 hit foundC45H69N7O3 - 1 hit found
Scan speed problem: Due to poor ion statistics only few scans are collectedMass accuracy and isotopic abundance accuracy are bad
Structural isomer lookup example in ChemSpider
In-silico fragmentation with MassFrontierusing fragmentation library of 20,000 mechanisms from literature
In-silico fragmentation with MassFrontier
C42H78NO8P – Fragments N.A.
Experimental peaks m/z 478.45 and 496.46 were detected in MS/MS spectrumIn-silico fragmentation should match the experimental fragmentation.In-silico - using a computer library of 20,000 fragmentation rules from the MS literature
C42H77NO10 – Fragments N.A. C45H69N7O3 – Fragments: m/z 496
C43H82NO7P – Fragments m/z 478 and 496LWHKEEJOPDYARM-WYCAKVPNBV
C43H82NO7PH – Fragments N.A.
Possible solution (2 fragments match)
Example discussion: Fragment at m/z 236 not explained; molecular ion may be wrong; Substance can be potential new compoundMust be confirmed by NMR or external standard
General problems: Best approach is to generate MS and MSn and MSe mass spectral librariesAdduct removal is a problemBuilding target lists is always good (know what to expect)Focus on certain substance classes onlyFocus on single compound only
Substance must be known for in-silico approachFragmentation rules must be captured for in-silico approachIn-silico approaches work best for peptides, carbohydrates, lipids(due to known and stable fragmentations)
Discussion of general LC-MS approach
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 generationMake use of high resolving power whenever possible
Use MS/MS data and mass spectra from different ionization voltagesUse existing MS/MS libraries or create your own MSn tree libraries
Use molecular isomer databases for obtaining possible structure candidatesConfirm if possible with MSn data or other possible filter constraints
Tasks (36 min):
(1) Download and install one tool from http://www.ms-utils.org/wiki/pmwiki.php/Main/SoftwareList
(2) Download sample data from Open Data repositories and report your experience with the selected software (1) on the sample data
Reading List (44 min):
Hardware and Software Challenges for the Near Future: Structure Elucidation Concepts via Hyphenated Chromatographic Techniques
QUALITATIVE AND QUANTITATIVE MASS SPECTRAL ANALYSIS
Congruent Strategies for Carbohydrate Sequencing. 3. OSCAR: An Algorithm for Assigning Oligosaccharide Topology from MSn Data
Data Reduction of Isotope-resolved LC-MS Spectra
Comparative LC-MS: A landscape of peaks and valleys
Links:
Used for research: (right click – open hyperlink)Mass Spectrometry in Life Science: Technology and Data-Evaluation
Managing and Exploring Large Data Sets Generated by Liquid Separation - Mass Spectrometry
Metabolomics Technologies applied to the Identification of Compounds in Plants
Of general importance for this course:http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/Structure_Elucidation/
top related