Welcome! Mass Spectrometry meets Cheminformatics Tobias Kind and Julie Leary UC Davis Course 7: Concepts for LC-MS Class website: CHE 241 - Spring 28 - CRN 16583 Slides: 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 MS n fragmentation 1 5 MS2 2 4 6
Deconvolution and evaluation of LC-MS data UPLC_C8_DataDependent_Chlamy_7373... 3/7/27 3:21:28 AM RT:. - 44.99 Relative Abundance 1 9 8 7 6 5 4 3 2 1 1.31 18.3 17.8 19.11 29.36 28.74 2.16 3.3 11.27 43.97 3.55 23. 17.49 23.8 16.7 5 1 15 2 25 3 35 4 Time (min) 43.68 9.55 1.56 6.37 31.53 37.19 43.9 UPLC_C8_DataDependent_Chlamy_73732128 #1414 RT: 18.3 AV: 1 NL: 8.23E5 F: FTMS + p NSI Full ms [2.-12.] 756.57745 1 R=5541 Relative Abundance 9 8 7 6 5 4 3 2 1 LC-MS Chromatogram Extracted Mass spectrum 754.57471 R=52 756.2686 R=87 755.5751 R=62 756.89417 R=866 757.5829 R=551 758.58636 R=561 757.927 R=792 759.59546 R=64 76.6144 R=522 NL: 1.68E6 m/z= 7.-2. F: FTMS + p NSI Full ms [2.-12.] MS UPLC_C8_DataDependent _Chlamy_73732128 761.59888 R=626 754 755 756 757 758 759 76 761 762 m/z Chromatogram Source: N.Saad, DY Lee FiehnLab Picture: nrel.gov 762.297 R=4991 LC-MS run 4 minutes C8 column, Agilent-UPLC Chlamydomonas extract + FT-ICR-MS mass spectrum MS1 @ 5, resolving power Check charge state = 1 756.57 represents [M+H] +
Peak Picking with ACD/SpecManager 9.
Processing of LC-MS data - use of MassFrontier
Deconvolution and evaluation of LC-MS data Example with HighChem Mass Frontier 1 75 LC-MS detected compound Marked with blue triangle 5 25 1. 15. 2. 25. 3. 35. 4. 45. LC-MS detected compound Marked with blue triangle 141 peaks extracted 1 756.577 75 5 25 76.568 237.733 324.29 436.437 516.755 585.457 85.99 871.588 951.566 117.9 1196.27 2 3 4 5 6 7 8 9 1 11 12 Extracted MS1 peak Library search useless (only single peak)
Adduct removal and detection during ESI-LC-MS runs 1 9 8 7 756.57745 R=5541 [M+H] + = 756.577 M = 755.5627 Relative Abundance 6 5 4 757.5829 R=551 3 2 1 758.58636 R=561 759.59546 R=64 756.5 757. 757.5 758. 758.5 759. 759.5 m/z Your M here: Your M+X or M-X 755.562724 756.577 Ion name Ion mass Charge Mult Mass Result: Reverse: 1. Positive ion mode M+2H M/2 + 1.7276 2+.5 1.7276 378.788638 377.277724 M+H+NH4 M/2 + 9.5255 2+.5 9.5255 387.31912 368.76445 M+H+Na M/2 + 11.998247 2+.5 11.998247 389.77969 366.286753 M+H M + 1.7276 1+ 1 1.7276 756.57 755.562724 M+NH4 M + 18.33823 1+ 1 18.33823 773.596547 738.536177 M+Na M + 22.989218 1+ 1 22.989218 778.551942 733.58782 2. Negative ion mode M-3H M/3-1.7276 3-.33-1.7276 25.846965 253.197276 M-2H M/2-1.7276 2-.5-1.7276 376.77486 379.292276 M-H2O-H M- 19.1839 1-1 -19.1839 736.544334 775.58839 M-H M - 1.7276 1-1 -1.7276 754.555448 757.577276 M+Na-2H M + 2.974666 1-1 2.974666 776.53739 735.595334 M+Cl M + 34.96942 1-1 34.96942 79.532126 721.6598 Download Adduct-Calculator [LINK]
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 4 other adducts? Example data from crocin standard mixture (expected MW: 976.965) 7-2-crocin-LTQFT-pos-V # 1 RT:. AV: 1 NL: 9.78E3 : FTMS + p NSI Full ms [ 5.-2.] 719.2179 1 95 27-2-crocin-LTQFT-pos-1V #1 RT:.1 AV: 1 NL: 1.3E4 T: FTMS + p NSI sid=1. Full ms [ 5.-15.] 1 95 999.37189 [M+Na] + 9 85 CID = V 9 85 CID = 1V 8 8 75 75 719.21747 7 7 65 65 6 6 Relative Abundance 55 5 45 529.17432 Relative Abundance 55 5 45 749.2296 4 35 367.12146 575.21149 4 35 135.36377 3 25 853.28149 115.33575 [M+Na] + 3 25 675.26556 529.17535 853.28583 2 2 367.12228 1177.39673 15 1 185.11496 31.14145 1177.3886 1241.42676 15 1 575.21246 5 1359.45837 1517.5293 2 4 6 8 1 12 14 16 m/z 1695.5 5 77.14542 329.95432 219.1861 485.2132 969.36615 1225.4128 1339.45142 2 4 6 8 1 12 14 m/z
Adduct removal and detection during LC-MS runs Your M here: Your M+X or M-X 853.3389 756.577 Ion name Ion mass Charge Mult Mass Result: Reverse: 1. Positive ion mode M+2H M/2 + 1.7276 2+.5 1.7276 427.672721 437.152724 M+H+NH4 M/2 + 9.5255 2+.5 9.5255 436.185995 428.63945 M+H+Na M/2 + 11.998247 2+.5 11.998247 438.663692 426.161753 M+H M + 1.7276 1+ 1 1.7276 854.338166 875.312724 M+NH4 M + 18.33823 1+ 1 18.33823 871.364713 858.286177 M+Na M + 22.989218 1+ 1 22.989218 876.3218 853.33782 2. Negative ion mode M-3H M/3-1.7276 3-.33-1.7276 251.182724 253.197276 M-2H M/2-1.7276 2-.5-1.7276 377.277724 379.292276 M-H2O-H M- 19.1839 1-1 -19.1839 737.55161 775.58839 M-H M - 1.7276 1-1 -1.7276 755.562724 757.577276 M+Na-2H M + 2.974666 1-1 2.974666 777.544666 735.595334 M+Cl M + 34.96942 1-1 34.96942 791.53942 721.6598 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 CHN 5 O 13 P 15 or CHN 11 P 19??? Apply Seven Golden Rules for correct molecular formulas Apply heuristic and mathematical and chemometric rules for filtering elemental compositions
Isotopic Pattern generator from formula Example from MWTWIN Is usually included in every LC-MS software
Isotopic pattern equally important as accurate mass Experimental result Abundances for all molecular formulae 1 756.57745 R=5541 25. 9 8 7 Relative Abundance 6 5 4 3 2 1 A+1 = 47.44% 757.5829 R=551 A+2 = 1.92 % 758.58636 R=561 759.59546 R=64 756.5 757. 757.5 758. 758.5 759. 759.5 m/z Isotopic Abundance A+2 [%] 2. 15. 1. 5... 2. 4. 6. 8. Isotopic Abundance A+1 [%] m/z Intensity Relative Resolution 756.5765 58934.1 1 5491 757.5814 279455.2 47.44 551 758.5862 64293.3 1.92 561 756.5526 19173.8 3.26 52 759.593 9656.4 1.64 56 We can discard all other results outside the error box. Current box reflect +/- 1% error.
Problems during LC-MS peak detection and MS deconvolution 48 36 Multiple peaks detected But it is a single component only 24 12 1.8 1.9 11. 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 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
UPLC-FT-MS data extraction with MassFrontier Mass Frontier 5. Report File MS 1 FTMS + p NSI Full #1414 MS1 1 756.577 5 Fragment peaks m/z 478.45 and 496.46 755 76 1 478.45 496.46 MS2 File MS 2 ITMS + c NSI d Full 756.58 #1415 1 478.45 496.46 5 335.56 2 4 6 75 5 25 236.2 MS2 271.6 335.56 391.34 434.52 687.72 25 3 35 4 45 5 55 6 65 7 1 75 5 756.577 MS1 25 751 752 753 754 755 756 757 758 759 76 761 762 Approach: generate molecular formula using Seven Golden Rules; find matching isomers in molecular databases; confirm possible matches by in-silico fragmentation (usually impossible);
Seven Golden Rules generate possible molecular formulas 5 formula candidates left with 3 ppm mass accuracy and 1% isotopic abundances These are candidates with good isotopic pattern match. These 5 were found in PubChem. C42H78NO8P - 1 isomer hit C42H77NO1-1 isomer hit C39H73N5O9 - 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
Structural isomer lookup example in ChemSpider
In-silico fragmentation with MassFrontier using fragmentation library of 2, mechanisms from literature
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 2, fragmentation rules from the MS literature C42H78NO8P Fragments N.A. C42H77NO1 Fragments N.A. C45H69N7O3 Fragments: m/z 496 C43H82NO7P Fragments m/z 478 and 496 LWHKEEJOPDYARM-WYCAKVPNBV C43H82NO7PH Fragments N.A. Possible solution (2 fragments match)
Discussion of general LC-MS approach Example discussion: General problems: Fragment at m/z 236 not explained; molecular ion may be wrong; Substance can be potential new compound Must be confirmed by NMR or external standard Best approach is to generate MS and MS n and MS e 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)
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
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/