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

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MS2

UPLC_C8_DataDependent_Chlamy_07030703... 3/7/2007 3:21:28 AM

RT: 0.00 - 44.99

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18.3017.80

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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

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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]+

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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

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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

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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

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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 ]

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7 1 9 .2 1 7 9 0

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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

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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]

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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

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Isotopic Abundance A+1 [%]

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756.5 757.0 757.5 758.0 758.5 759.0 759.5m/z

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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

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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

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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

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478.45496.46

236.20

434.52391.34 687.72335.56271.06

Mass Frontier 5.0 Report

File MS1 FTMS + p NSI Full #1414

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756.577

File MS 2 ITMS + c NSI d Full 756.58 #1415

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478.45 496.46

335.56

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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/