Fourier Transform Ion Cyclotron Resonance Mass Spectrometer In Metabolomics. FT-ICR MS or FTMS

Transcription

1 Fourier Transform Ion Cyclotron Resonance Mass Spectrometer In Metabolomics FT-ICR MS or FTMS

2 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry - Will determain the m/z ratio of an ion - Ions are formed as in other mass analysers: e.g. Electrospray Ionization (ESI); Matrix Assisted laser Desorption Ionization (MALDI); Electron Ionization (EI); Chemical Ionization (CI)

3 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry - The m/z ratio measurment of an ion is based upon the ion's motion or cyclotron frequency in a magnetic field - Ions are detected by passing near detection plates and thus differently from other mass detectors/analysers in which ions are hitting a detector (at diferent times or places)

4 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry - The ions are trapped in a magnetic field combined with electric field perpendicular to each other (Penning trap) - They are excited to perform a cyclotron motion - The cyclotron frequency depends on the ratio of electric charge to mass (m/z) and strength of the magnetic field

5 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

6 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry - This frequency signal "image" (Ion Cyclotron Resonance, ICR signal) is detected by a pair of opposing electrode plates

7 f = ion cyclotron frequency in khz B = is the magnetic field strength in Tesla z = is the charge state of the ion M = is the mass of the ion in Da. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry - The ICR signal is converted to a frequency spectrum by Fourier transformation - The relation between the cyclotron frequency and the m/z could be simply presented as: f = Bz/m

8 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry The frequency spectrum is converted to a mass spectrum ICR signal of each ion (a single m/z)

9 FTMS Characteristcs - The highest resolving power of all mass analyzers - The highest mass accuracy- usually around 1ppm or less Ability of a mass analyzer to assign the mass of an ion close to its true value (exact mass) m accuracy = mreal - mmeasured In ppm = 10 6 * m accuracy / mmeasured

10 FTMS Characteristcs - Similar sensitivity to Q-TOF - Limit of detection in MS/MS mode lower than for Q-TOF (Q-FTMS will be better) -MS n could be performed - Most ionization modes could be used

11 FTMS metabolic phenotyping Crude biological extract Direct injection into FTMS via HPLC autosampler +/- ESI and +/- APCI ionization High resolution separation and detection of metabolites Instrument acquisition time: (2-5 min/sample/mode) Off-line spectral processing Exportation of processed data to database

12 Analytical Instrumentation (FTMS) Fourier Transform Ion Cyclotron Mass Spectrometry

13 Non-targeted metabolic profiling in strawberry Green Red White Turning

14 Positive ion ESI of strawberry fruit Red Strawberry Green Strawberry 800 amu Window

15 High resolution separation X = Y = X m/z X Y Resolution = 135,000, 0.1 AMU Window

16 Empirical formula determination Internal Mass Calibration Of Sample Calibration Results: Ref. Masses Exp. Masses Diff (ppm) Calculate Accurate Mass Of Metabolite Assign Empirical Formula Based Upon Calibration Errors Calculate Possible Empirical Formulas Mass Analysis for mass # 12C 1H 16O 14N 32S mass error = C 21 H 20 O 10 [+H] + Error = 0.52ppm e e e e e e e e e e e e e e e e e e e e e e e e e e e-06

17 Number of mass peaks and metabolites observed Number of metabolites observed across stages of fruit development, ionization modes and extractions Overall mass peaks Unique mass peaks Green White Turning Red Total Only Both (%) Total 50 AN 50 AN 50 AN 50 AN 50 + AN 50 AN 50 + AN ESI (25%) 1472 ESI (16%) 1270 APCI (36%) 1499 APCI (19%) 1603 Mean (24%) 1461 Total , (24%) 5844

18 Accurate empirical formula determination 100% Empirical Formula Assignment Statistics 80% 60% 40% 20% 0% ESI + ESI - APCI + APCI - Only 1 Formula 2 Formulas More than 2

19 Quantitative determination of change between samples Metabolite Changes Observed In Strawberry Development 1500 RS/GS RS/WS 2X 2X RS/TS 1000 TS/GS 2X TS/WS X WS/GS 5X 10X 2X 5X 10X 5X 10X 2X 5X 10X 5X 10X 5X 10X Metabolite Increases Metabolite Decreases

20 Metabolic profiling of strawberry fruit development Metabolite CG EF NM IM EX R/G T/G W/G Group 1: Early expression Profile A Ellagic acid* P. Acid C 14 H 6 O AP- AN Hydroxybenzoic acid* P. Acid C 7 H 6 O AP- AN Arginine A. acid C 6 H 14 N 4 O ES Vanillic acid* P. Acid C 8 H 8 O AP- AN Malic acid O. acid C 4 H 6 O ES- AN Catechin (epi)/leucopelargonidin* Phenolic C 15 H 14 O ES Caffeate* P. Acid C 9 H 8 O AP Ethyl cinnamate* Ester C 11 H 12 O AP- AN Quercitin glucoside* Flavonoid C 21 H 20 O ES Methyl cinnamate* Ester C 10 H 10 O AP- AN Dihydrokaempferol* Flavonoid C 15 H 12 O AP Leucocyanidin* Flavonoid C 15 H 14 O AP- AN N-Alanylcysteine A. Acid d. C 6 H 12 N 2 O 3 S ES+ AN Gentisic/Protocatechuic acid* P. Acid C 7 H 6 O AP Malusfuran Furan C 12 H 8 O AP (E)2-hexenol / hexanal Alcohol C 6 H 12 O AP+ AN Glucogallin* Phenolic C 13 H 16 O AP Pantothenic acid Vitamin C 9 H 17 NO ES+ AN Dihydroxybenzoquinone Phenolic C 6 H 4 O ES+ AN Dihydroquercitin* Flavonoid C 15 H 12 O AP

21 Metabolic profiling of strawberry fruit development Metabolite CG EF NM IM EX R/G T/G W/G Profile I Linoleic acid F. Acid C 18 H 32 O AP+ AN Palmitoleic acid F. Acid C 16 H 30 O AP+ AN Oleic acid F. Acid C 18 H 34 O AP+ AN Pentadecanoic acid F. Acid C 15 H 30 O AP+ AN Palmitic acid F. Acid C 16 H 32 O AP+ AN methylbutyl nonanoate Ester C 14 H 28 O AP+ AN Stearic acid F. acid C 18 H 36 O AP+ AN Furaneol (R=D-glucopyranose) Furan C 12 H 18 O ES+ AN Furaneol (R=D-glucopyranose, malonyl) Furan C 15 H 20 O ES Nonanoic acid F. Acid C 9 H 18 O AP+ AN Capric acid F. Acid C 10 H 20 O AP+ AN Eicosanoic acid F. Acid C 20 H 40 O AP+ AN Linolenic acid F. Acid C 18 H 30 O AP+ AN Cinnamate* P. Acid C 9 H 8 O AP octyl butanoate Ester C 12 H 24 O AP+ AN methyhexyl hexanoate Ester C 13 H 26 O AP+ AN Cis-hex-3-enyl octanoate Ester C 14 H 26 O AP+ AN Methylstearate Ester C 19 H 38 O AP+ AN Sinapyl alcohol* Phenolic C 11 H 14 O AP- AN Scopoletin* Benzopyranoid C 10 H 8 O AP Isopropyl-3-methoxypyrazine Alkaloid C 8 H 12 N 2 O AP+ AN Farnesyl acetone Terpene C 18 H 30 O AP+ AN Hydroxycoumarine* Benzopyranoid C 9 H 6 O AP+ AN Coumarine* Benzopyranoid C 9 H 6 O AP+ AN Pelargonidin* Flavonoid C 15 H 10 O ES Smipine Alkaloid C 10 H 16 N 2 O AP+ AN

22 Group 1: Early expression Catechin (APCI +) Heptylamine (ESI+) Citrate/Isocitrate (APCI+)

23 Group 2: Intermediate expression Phenylalanine (ESI+) Glutamine (ESI+) Benzoate (APCI+)

24 Group 3: Late expression Pelargonidin glucoside (ESI+) Coumaroyl glucoside (ESI-) Furaneol glucoside (ESI+)

25 Glucogallin (hydrolyzable tannins)(-4.3) Gallic acid (+2.0) Ellagic acid (-33.3) Hydroxycoumarin (+5.44) Quercetin Quercetin glucoside (-7.7) Catechol (+2.0) Protocatechoic acid (-3.2) Vanillic acid (-12.5) Kaempferol glucoside (+2.1) Dihydroquercetin (-2.4) DFR (1.9) Leucocyanidin (-2.7) Cyanidin ANS (+6.7) GT1 (+8.3) Cyanidin glucoside (+2.1) GST (+2.8) Anthranilate (-2.3) Coumarin (+5.5) Hydroxybenzoic acid (-11.1) Kaempferol Catechin (condensed tannins) (-25) vacuole Coumarate glucose (+9.6) Gentisic acid (-3.2) Benzoic acid (+2.0) CHS (+3.3) Naringenin chalcone CHI (+4.3) Naringenin F3H (+3.2) Dihydrokaempferol (-9.1) DFR (1.9) Leucopelargonidin (-25) ANS (+6.7) Pelargonidin (+9.46) GT1 (+8.3) Pelargonidin glucoside (+255) GST (+2.8) Phenylalanine (-5.0) PAL Cinnamate (+5.8) Coumarate (+3.2) Coumaroyl - CoA CCR (+3.6) GT2 (+3.7) Caffeate Ferulate (+3.9) Hydroxyferulate (+2.4) Sinapate (+3.8) Sinapoyl CoA OMT (+2.6) OMT (+2.6) Caffeoyl CoA Scopoletin (+5.31) CCR (+3.6) Cinnamate glucose (+2.0) Ethyl cinnamate (-7.7) Methyl cinnamate (-5.3) + Quinic acid (-4.2) Chlorogenic acid Feruloyl CoA Coniferaldehyde Coniferyl alcohol CCR (+3.6) CAD (+8.8) Sinapaldehyde (+2.4) CAD (+8.8) Sinapyl alcohol CAD (+5.8) Coumaraldehyde (+8.8) (+5.86) Coumaryl alcohol (+3.29)

26 Phenylalanine and changes in gene and metabolite expression?

27 Glucogallin (hydrolyzable tannins)(-4.3) Gallic acid (+2.0) Ellagic acid (-33.3) Hydroxycoumarin (+5.44) Quercetin Quercetin glucoside (-7.7) Catechol (+2.0) Protocatechoic acid (-3.2) Vanillic acid (-12.5) Kaempferol glucoside (+2.1) Dihydroquercetin (-2.4) Cyanidin DFR (1.9) Leucocyanidin (-2.7) ANS (+6.7) GT1 (+8.3) Cyanidin glucoside (+2.1) GST (+2.8) Anthranilate (-2.3) Coumarin (+5.5) Hydroxybenzoic acid (-11.1) Kaempferol Catechin (condensed tannins) (-25) vacuole Coumarate glucose (+9.6) Gentisic acid (-3.2) Benzoic acid (+2.0) CHS (+3.3) Naringenin chalcone CHI (+4.3) Naringenin F3H (+3.2) Dihydrokaempferol (-9.1) DFR (1.9) Leucopelargonidin (-25) ANS (+6.7) Pelargonidin (+9.46) GT1 (+8.3) Pelargonidin glucoside (+255) GST (+2.8) Phenylalanine (-5.0) PAL Cinnamate (+5.8) Coumarate (+3.2) Coumaroyl - CoA CCR (+3.6) GT2 (+3.7) Caffeate Ferulate (+3.9) Hydroxyferulate (+2.4) Sinapate (+3.8) Sinapoyl CoA OMT (+2.6) OMT (+2.6) Caffeoyl CoA Scopoletin (+5.31) CCR (+3.6) Cinnamate glucose (+2.0) Ethyl cinnamate (-7.7) Methyl cinnamate (-5.3) + Quinic acid (-4.2) Chlorogenic acid Feruloyl CoA Coniferaldehyde Coniferyl alcohol CCR (+3.6) CAD (+8.8) Sinapaldehyde (+2.4) CAD (+8.8) Sinapyl alcohol CAD (+5.8) Coumaraldehyde (+8.8) (+5.86) Coumaryl alcohol (+3.29)

28 Phenylalanine and changes in gene and metabolite expression?

29 Glucogallin (hydrolyzable tannins)(-4.3) Gallic acid (+2.0) Ellagic acid (-33.3) Hydroxycoumarin (+5.44) Quercetin Quercetin glucoside (-7.7) Catechol (+2.0) Protocatechoic acid (-3.2) Vanillic acid (-12.5) Kaempferol glucoside (+2.1) Dihydroquercetin (-2.4) Cyanidin DFR (1.9) Leucocyanidin (-2.7) Coumarin (+5.5) ANS (+6.7) GT1 (+8.3) Cyanidin glucoside (+2.1) GST (+2.8) Anthranilate (-2.3) Hydroxybenzoic acid (-11.1) Kaempferol Catechin (condensed tannins) (-25) vacuole Coumarate glucose (+9.6) Gentisic acid (-3.2) Benzoic acid (+2.0) CHS (+3.3) Naringenin chalcone Naringenin F3H (+3.2) Dihydrokaempferol (-9.1) DFR (1.9) Leucopelargonidin (-25) ANS (+6.7) Pelargonidin (+9.46) GT1 (+8.3) Pelargonidin glucoside (+255) GST (+2.8) CHI (+4.3) Phenylalanine (-5.0) PAL Cinnamate (+5.8) Coumarate (+3.2) Coumaroyl - CoA CCR (+3.6) GT2 (+3.7) Caffeate Ferulate (+3.9) Hydroxyferulate (+2.4) Sinapate (+3.8) Sinapoyl CoA OMT (+2.6) Caffeoyl CoA Scopoletin (+5.31) OMT (+2.6) CCR (+3.6) Cinnamate glucose (+2.0) Ethyl cinnamate (-7.7) Methyl cinnamate (-5.3) + Quinic acid (-4.2) Chlorogenic acid Feruloyl CoA Coniferaldehyde Coniferyl alcohol CCR (+3.6) CAD (+8.8) Sinapaldehyde (+2.4) CAD (+8.8) Sinapyl alcohol CAD (+5.8) Coumaraldehyde (+8.8) (+5.86) Coumaryl alcohol (+3.29)

30 Over-expression of FaMYB1 in Tobacco Tr-m Tr-w Tr-bcred Tr-bcwhite Tr-s Nt A B C D

31 The phenylpropanoid pathway in strawberry & tobacco H O Lignin Phenylalanine Kaempferol Quercetin Dihydroquercetin OH O + FLS Cyanidin OH CAD DFR ANS OH OH PAL C4H 4 - Coumarate 4 - Coumaroyl - CoA Malonyl - CoA FLS F3 H Cinnamate 4CL CHS Naringenin chalcone HO CHI Naringenin F3H Dihydrokaempferol OH O + DFR ANS Pelargonidin OH OH GT Cyanidin -3-glucoside (tobacco) GT Pelargonidin -3-glucoside (strawberry) RT GST GST vacuole

32 Differentially Expressed Masses Between Wild- Type and Transgenic Flowers No. IM OM T1-white Bc-red Bc-white 1 APCI a = 2 APCI = 3 APCI = 4 APCI = 5 APCI = 6 APCI = 7 APCI = 8 ESI = 9 ESI =

33 Wild type vs. White Transgenic Tobacco (mass no. 9) Observed mass of unknown: Empirical formula search result (1 possibility only): C 27 H 30 O 15 [+H]+ Mass: m/z Mass error: 0.04 ppm

34 18 Possibilities in Search of the Natural Product Database for C27H30O15: Name Keracyanin, CI, INN8 3,3',4',5,7-Pentahydroxyflavylium(1+); 3-glucopyranoside-D-β-O-5Rhamnoside, -L-α-O 3,3',4',5,7-Pentahydroxyflavylium(1+); 3-]galactopyranoside-D- (4 1)-Rhamnopyranosyl-L-α]-O 3,3',4',5,7-Pentahydroxyflavylium(1+); 3-]galactopyranoside-D-β- (6 1)-Rhamnopyranosyl-L-α]-O 3,3',4',5,7-Pentahydroxyflavylium(1+); 3-]glucopyranoside-D-β- (2 1)-Rhamnopyranosyl-L-α]-O 3,4',5,6,7-Pentahydroxyflavylium(1+); 3-]glucopyranoside-D-β- (6 1)-Rhamnopyranosyl-L-α]-O 3,4',5,7-Tetrahydroxyflavylium(1+), glucopyranoside-d-β-o-di-3,5 ;CI8 3,4',5,7-Tetrahydroxyflavylium(1+), CI8; 3- ]glucopyranoside-d-β-(2 1)-Glucopyranosyl-D-β]-O 3,4',5,7-Tetrahydroxyflavylium(1+), CI8; 3- ]glucopyranoside-d-β-(6 1)-Glucopyranosyl-D-β]-O 3',4',5,7-Tetrahydroxyflavylium(1+); 5,7-Di-glucopyranoside-D-β-O CAS Registry Number Molecular Formula C 27 H 31 O 15 (+) C 27 H 31 O 15 (+) C 27 H 31 O 15 (+) C 27 H 31 O 15 (+) C 27 H 31 O 15 (+) C 27 H 31 O 15 (+) C 27 H 31 O 15 (+) C 27 H 31 O 15 (+) C 27 H 31 O 15 (+) C 27 H 31 O 15 (+)

35 All 18 Possibilities for C27H30O15 are glycosides of either Cyandin, Pelargonidin, Peonidin or Luteolidin One of the candidates, Keracyanin (cyanidn-3- rhamnoside) was reported in 1992 to accmulate in Tobbaco flowers.

36 Entry Name: Keracyanin, 8CI, INN Synonym(s): 3-O- Rhamnoglucosyloxycyanidin. Ceracyanin. Sambucin. Antirrhinin. Cyanidin 3- rutinoside. Cyaninoside. Meralop. Noctilux. Nyctalux. Prunicyanin. Oleocyanin Chapman & Hall Number: BFH51-Q CAS Registry Number: Type of Compound Code(s): VK0050 Molecular Formula: C 27 H 31 O 15 (+) Molecular Weight: Accurate Mass: Percentage Composition: C 54.45%; H 5.25%; O 40.30% General Statement: Glycoside of 3,3',4',5,7-Pentahydroxyflavylium(1+) CMQ73-Z Biological Source: Pigment present in fresh blossoms of Canna generalis and Silene dioica, as well as in Prunus, Viola, Tulipa and other plants Biological Use/Importance: Improves vision in humans (for night blindness) Physical Description: Fine red needles (dil. HCl) (as chloride) Melting Point: Mp 175 (chloride) Other Data: CAS no. refers to chloride

37 Confirmation of Cyandin 3- Rhamnoglucoside in Mass no. 9)

38 Enhancing the Data Obtained by High Resolution MS Using Isotope Abundune

39 Using Abundance of Isotopes for Metabolite Elucidation (in Spectra)

40 Metabolite Annotation Scheme e.g. MOLGEN

41 Molecular Formula Search in Different Databases

42 Lower Mass Accuracy with Good Isotope Abundunce Analysis Compared to Higher Mass Accuracy and no Isotope Analysis

43 The Conclusion from the Study A Mass spectrometer capable of 3ppm mass accuracy and 2% error for isotopic abundunce patterns outperforms mass spectrometers with less than 1ppm mass accuaracy that does not include isotope information in the calculation of molecular formulae

44 Silylated Sorbitol (5% error on isotopic abundances)

45 Full spectrum of Rutin standard M+H

46 Zoom on the Isotope pattern of

47 Elemental composition analysis of

48 Isotope pattern matching of Calculated isotopic pattern For C27H31O16 By the program measured isotopic pattern measured

49 See You Next Week