RmassBank Decelopments: XCMS+CAMERA=MetShot Steffen Neumann, Erik Müller Leibniz Institute of Plant Biochemistry ThOB 13.6.2013 S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Metabolomics – The Pipeline S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Outline 1 Acquisition and Processing of LC-MS/MS 2 Summary & Outlook S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Outline 1 Acquisition and Processing of LC-MS/MS 2 Summary & Outlook S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Instrument Data-dependent MS/MS Instrument Auto-MS/MS Full scan first Then: MS/MS of most intense 800 peak(s) Online 600 m/z Problems: 400 Boring peaks: plasticiser, solvent, . 200 Molecular ion / Biologically relevant peaks may not be 0 500 1000 1500 2000 the most intense RT DDIT Noise spectra / Too few / too precursor selection in 8 LC-MS/MS runs many peaks S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Instrument Data-dependent MS/MS Instrument Auto-MS/MS Full scan first Then: MS/MS of most intense peak(s) Online Problems: Boring peaks: plasticiser, solvent, . Molecular ion / Biologically relevant peaks may not be the most intense Noise spectra / Too few / too many peaks S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Instrument Data-dependent MS/MS Instrument Auto-MS/MS Full scan first Then: MS/MS of most intense peak(s) Online Problems: Boring peaks: plasticiser, solvent, . Molecular ion / Biologically relevant peaks may not be the most intense Noise spectra / Too few / too many peaks S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Instrument Data-dependent MS/MS Instrument Auto-MS/MS Full scan first Then: MS/MS of most intense peak(s) Online Problems: Boring peaks: plasticiser, solvent, . Molecular ion / Biologically relevant peaks may not be the most intense Noise spectra / Too few / too many peaks S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Single spectrum extraction m/z accuracy: Low intensity signals ! low m/z accuracy ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● 285.080 20 ppm ● ● ● ●●● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● m/z ● ● ● ● Spectra merging ● ● ! improves m/z accuracy 285.065 530 535 540 545 550 ●● ● ● Co-elution / co-isolation: ● ● ● ● ● ● 60000 MS/MS spectrum contains ● ● Intensity ● ● ● ● ● 20000 ● fragment peaks from more ● ● ● ●● ● ●●● ●●●●●●●●● ●●●●●●●●● ● 0 than one metabolite 530 535 540 545 550 Seconds Poor identification S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Single spectrum extraction m/z accuracy: Low intensity signals 50 ! low m/z accuracy Counts 95 40 89 Spectra merging 83 77 ! improves m/z accuracy 30 72 66 60 54 Co-elution / co-isolation: dppm 20 48 42 MS/MS spectrum contains 36 30 10 24 fragment peaks from more 19 13 than one metabolite 7 0 1 Poor identification 4 6 8 10 12 14 log(intensity) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Acquisition of LC-MS/MS Manual LC-MS/MS acquisition: Data-dependent MS/MS: Choose m/z – RT window(s) Selects most intense peak(s) ! build instrument method(s) Low quality spectra: 2nd run/injection for MS/MS “wrong” precursor(s) Manual extraction m/z accuracy effective MS1 scan rate of MS/MS spectrum reduced Controlled quality data Online, little control Offline, low throughput Choice of two approaches with different drawbacks MetShot is a hybrid approach, where the instrument methods are created automatically after the profiling and statistics S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 MetShot: nearline data-dependent tandem MS Peaks of Interest Library Search LC/MS data MS/MS data de-Novo Identification XCMS MS/MS Method XCMS centWave Generation Peak Table Peak Tables Statistics CAMERA CAMERA MS/MS Spectra Steffen Neumann, Andrea Thum and Christoph Böttcher: Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data. Metabolomics (2012) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Scheduling of MS/MS spectra MSMSpos_Col0_1_10eV_1−A,1_01_9844 “Features of Interest” list from LC-MS profiling 800 Optimal packing of LC-MS/MS: 600 m/z most important first without RT overlap 400 method files for Bruker, Waters, Agilent, TraML XML 200 Ordered by p-Value 0 200 400 600 800 (biological relevance) Seconds ) Only few LC-MS/MS runs to cover many peaks R package: http://msbi.ipb-halle.de/msbi/MetShot Steffen Neumann, Andrea Thum and Christoph Böttcher: Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data. Metabolomics (2012) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Scheduling of MS/MS spectra “Features of Interest” list from LC-MS profiling 800 Optimal packing of LC-MS/MS: 600 m/z most important first without RT overlap 400 method files for Bruker, Waters, Agilent, TraML XML 200 Ordered by p-Value 0 200 400 600 800 (biological relevance) RT ) Only few LC-MS/MS runs to cover many peaks R package: http://msbi.ipb-halle.de/msbi/MetShot Steffen Neumann, Andrea Thum and Christoph Böttcher: Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data. Metabolomics (2012) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 MetShot: nearline data-dependent tandem MS Peaks of Interest Library Search LC/MS data MS/MS data de-Novo Identification XCMS MS/MS Method XCMS centWave Generation Peak Table Peak Tables Statistics CAMERA CAMERA MS/MS Spectra Steffen Neumann, Andrea Thum and Christoph Böttcher: Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data. Metabolomics (2012) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Collect and Assemble LC-MS and LC-MS/MS Feature detection with XCMS in LC-MS/MS raw data Collect compound spectra with CAMERA Assign spectra to nearest precursor peak of interest Steffen Neumann, Andrea Thum and Christoph Böttcher: Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data. Metabolomics (2012) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Evaluation of extracted spectra Xanthohumol 15 eV Xanthotoxin 15 eV Sample Sample PB005728 PB006163 Mixture of 27 standards Manual spectra extraction Intensity with vendor software, Intensity available in MassBank MassBank spectral score automatic vs. manual −1000 −500 0 500 1000 −1000 −500 0 500 1000 200 250Score >0.95 300 for 350 25 of 27 120 140 160 180 200 220 mz mz Naringin 15 eV Bergapten 15 eV Sample Sample PB005723 PB006144 Steffen Neumann, Andrea Thum and Christoph Böttcher: Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data. Metabolomics (2012) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Intensity Intensity −1000 −500 0 500 1000 −1000 −500 0 500 1000 150 200 250 300 350 400 120 140 160 180 200 220 mz mz Rotenone 15 eV Cinchonine 15 eV Sample Sample PB005742 PB005842 Intensity Intensity −1000 −500 0 500 1000 −1000 −500 0 500 1000 150 200 250 300 350 400 150 200 250 300 mz mz Anisic acid 15 eV Erythromycin 15 eV Sample Sample PB006062 PB005802 Intensity Intensity −1000 −500 0 500 1000 −1000 −500 0 500 1000 120 125 130 135 200 300 400 500 600 700 mz mz Evaluation of extracted spectra Xanthohumol 15 eV Xanthotoxin 15 eV Sample Sample PB005728 PB006163 Mixture of 27 standards Manual spectra extraction Intensity with vendor software, Intensity available in MassBank MassBank spectral score automatic vs. manual −1000 −500 0 500 1000 −1000 −500 0 500 1000 200 250Score >0.95 300 for 350 25 of 27 120 140 160 180 200 220 mz mz Naringin 15 eV Bergapten 15 eV Sample Sample PB005723 PB006144 Steffen Neumann, Andrea Thum and Christoph Böttcher: Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data. Metabolomics (2012) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 Intensity Intensity −1000 −500 0 500 1000 −1000 −500 0 500 1000 150 200 250 300 350 400 120 140 160 180 200 220 mz mz Rotenone 15 eV Cinchonine 15 eV Sample Sample PB005742 PB005842 Intensity Intensity −1000 −500 0 500 1000 −1000 −500 0 500 1000 150 200 250 300 350 400 150 200 250 300 mz mz Anisic acid 15 eV Erythromycin 15 eV Sample Sample PB006062 PB005802 Intensity Intensity −1000 −500 0 500 1000 −1000 −500 0 500 1000 120 125 130 135 200 300 400 500 600 700 mz mz S4: Tandem Mass Spectra of Natural Product Standards Table 2: Similarity scores between the automatically extracted tandem mass spectra Evaluation of extracted spectraand the reference spectra deposited in MassBank. The similarity score is based on the modified cosine distance used in the MassBank system. Sheet2 Compound Name MassBank Score Anisic acid PB006062 0.9981 Xanthotoxin PB006163 0.9966 Genistein PB005713 0.9953 Amentoflavone PB006304 0.9934 Xanthohumol PB005728 0.9919 Biochanin A PB005708 0.9906 Cinchonine PB005842 0.9902 Mixture of 27 standards Kaempferol PB005703 0.9889 4-Methylumbelliferyl glucoronide PB006265 0.9888 Manual spectra extraction Bicuculline PB005942 0.9882 Chlorogenic Acid PB006181 0.9872 with vendor software, Laudanosine PB005881 0.9864 (+/-) Salsolinol PB006041 0.9861 available in MassBank Rotenone PB005742 0.9849 Naringin PB005723 0.9832 MassBank spectral score Indole-3-acetyl-L-valine PB005789 0.9831 Rutin PB006202 0.9828 automatic vs. manual Indole-3-carboxylic acid PB005786 0.9803 Reserpine PB005761 0.9789 Hesperidin methyl chalcone PB006242 0.9771 Score >0.95 for 25 of 27 Chelidonine PB005902 0.9740 Erythromycin PB005802 0.9692 3,4,5-Trimethoxycinnamic acid PB005822 0.9679 Quercetin PB006205 0.9654 Bergapten PB006144 0.9527 Indole-3-acetonitrile PB006045 0.9440 Emetine PB005910 0.8247 Steffen Neumann, Andrea Thum and Christoph Böttcher: Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data. Metabolomics (2012) S. Neumann (IPB-Halle.DE) RMassBank ThOB 13.6.2013 5 Page 5 MetShot: nearline data-dependent tandem MS Peaks of Interest Library Search LC/MS data MS/MS data de-Novo Identification XCMS MS/MS Method XCMS centWave Generation Peak Table Peak Tables Statistics CAMERA CAMERA MS/MS Spectra S.