High Resolution LC-MS Data Output and Analysis · Glucose Quercetin. Spectra Components ... Rutin +...
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Transcript of High Resolution LC-MS Data Output and Analysis · Glucose Quercetin. Spectra Components ... Rutin +...
High Resolution LCHigh Resolution LC--MS MS Data Output and AnalysisData Output and Analysis
LC/MS
Sample
Introduction
Sample
Preparation
IonsDetection
IonsSeparation
IonizationMS Interface
UVSpectra
Separation
(column)
Efficient
Gradient
Your Sample
Result
LC-MS
Data (computer)Today
Mass SpectrometerMass SpectrometerMass Spectrometer
1. Breaks up constituents into molecular ions and other fragments
2. The ions are separated according to their mass-to-charge ratio (m/z)
3. Measures masses
Mass SpectrometerMass SpectrometerMass Spectrometer
4. Structural information on metabolite
* fragmentation pattern
* accurate mass
Alternative Ionization ModesAlternative Ionization Modes
•Atmospheric Pressure Ionization (API), in LC-MS
• Electrospray Ionisation (ESI): polar and semi-polar
• Atmospheric Pressure Chemical Ionization (APCI): less polar
ESAPCI
lipids waterpolarity of analyte molecule
Positive or Negative Modes?Positive or Negative Modes?
The formation of positive or negative ions depends on the sign of the applied electrical field
ES+: (M+H)+
Good ionization of basic compounds (get proton)E.g. amino, amide, ester, aldehyde/keto functional groups (formic acid in sample solution to help ionize)
ES-: (M-H)-
Acidic Compounds (give proton) E.g. organic acids, containing OH (ammonium buffer in sample solution to help ionize)
The formation of positive or negative ions depends on the sign of the applied electrical field
ES+: (M+H)+
Good ionization of basic compounds (get proton)E.g. amino, amide, ester, aldehyde/keto functional groups (formic acid in sample solution to help ionize)
ES-: (M-H)-
Acidic Compounds (give proton) E.g. organic acids, containing OH (ammonium buffer in sample solution to help ionize)
Mass AnalyzersMass Analyzers•Ion Cyclotron(FT-ICR-MS)
•Time of Flight(TOF)
•Magnetic Sector
•Quadrupole Ion Trap
•Quadrupole
“High Resolution”Instruments
“Low Resolution”Instruments
Mass AnalyzersMass AnalyzersMass
Accuracy
<1ppm•Ion Cyclotron(FT-ICR-MS)
•Time of Flight(TOF)
3-10ppm
2-5ppm•Magnetic Sector
•Quadrupole Ion Trap
•Quadrupole
n/a
n/a
LC-PDA-QTOF-MS of a Tomato Sample
MS-sample - Total Ion Chromatogram
(TIC)
MS-reference (lock mass) injected every few scans (TIC)
PDA-sample
Spectra Obtained at Each Scan
MS-sample
MS-reference (lock mass)
PDA-sample
Enkephalin (556.2766 in ES+)
Extracted chromatogram of a Specific Ion
TIC
Extracted chromatogramat m/z 611
Extracted chromatogramat m/z 303
LC/UV/MS Chromatograms of Standard’s Mixture(UV at 240nm, ES+ and ES-)
1 – Chlorogenic acid2 – Caffeic acid3 – Rutin4 – p-Coumaric5 – Quercetin-3-glucoside6 – Ferulic acid7 – Sinapic acid8 – Benzoic acid9 – Morin (Internal Standard)10 – Tomatin11 – Quercetin12 – Cinnamic acid13 – Naringenin Chalcone14 – Naringenin15 – Kaempferol
MS (-)
09-Jan-2006Standard Mix
Time1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
%
17
1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
AU
0.0
2.0e-1
4.0e-1
6.0e-1
8.0e-1
MixAllStand_b1 3: Diode Array Range: 1.113.68
3.63
2.67
2.26
1.66
3.38
3.26
5.43
4.954.606.136.00
5.92
MixAllStand_b1 1: TOF MS ES+ TIC
1.18e45.16
3.653.502.28
1.69
3.282.69
3.71 4.98
4.54
6.02
5.455.93
6.10
6.46
1
2
3
3
4
5
6
6
7
89
11
12
1314
15
12
45 7 9
10
1112
1314
15
UV
MS (+)
10-Jan-2006
Tim e1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
%
9
ILMixAllStand-c4 1: TOF MS ES- T IC
8.88e36.07
5.99
3.372.68
2.272.22
3.25
5.42
4.92
3.68
4.58
3.95
5.91
6.13
6.376.72 6.77
1
2 4
56
9
11
12
13
14
15
3 7
8
In the Spectra: Molecular Ion- An unfragmented (parent) ion formed by loss or gain of an proton from a molecule
- Has the same mass as the metabolite being analyzed (+ H or - H)
The Molecular Ion
Chromatogram and Spectrum of Morin
ES(+)
ES(-) [M-H]-
[M+H]+
Chromatograms in ES(+) & ES(-) modes
Spectra'sin ES(+) & ES(-) modes
Adduct Formation in Spectra (Na)
[M+H]+
[M+Na]+
Rutin C27H30O16
Na (22)
C27H31O16
C27H30O16Na
S5
Typical Adducts formed in LCMSTypical Adducts formed in LCMS
Mass of Adduct IonSource of AdductAdducts Ion
M+59Acetic acid[ M+CH3COO ]-
M+35Chlorinated solvent[ M+Cl ] -
M+18Ammonia[ M+NH4 ] +
M+23Sodium Salt[ M+Na ]+
M+39Potassium Salt[ M+K ]+
M+42Acetonitrile[ M+CH3CNH ]+
M+33Methanol[ M+CH3OHH ]+
M+19Water[ M+H2O]+
Isotopes in Spectra (13C)
[M+H]+
[M+Na]+Rutin C27H30O16
C27H31O16
C27H30O16Na
13C (little from other isotopes H, O)
Abundance of Isotopes (in Spectra)
An example of how isotopes can aid An example of how isotopes can aid in peak in peak identificationidentification
A (50.6%) A + 2 (49.3%)
The ratio of peaks containing 79 Br and its isotope 81 Br (100/98) confirms the presence of bromine in the compound.
Fragments of a Metabolite in Spectra
[M+H]+
[M+Na]+
Glucose (162)
Rhamnose (146)
MS spectrum of Rutin in ES(+)
Rhamnose
Glucose
Quercetin
Spectra ComponentsSpectra Components
Molecular IonMolecular IonFragments of MetaboliteFragments of MetaboliteAdducts (from solvent or diAdducts (from solvent or di--tritri--mers)mers)IsotopesIsotopes
Spectrum of Quercetin Trisaccharide(a Rutin Derivative)
Rutin
Quercetin[M+H]+
[M+Na]+
m/z100 200 300 400 500 600 700 800 900 1000 1100
%
0
100 765.1812
743.1995
303.0515
76.0834
469.1320391.1980 611.1645
470.1434
471.1271
612.1766
613.1899
766.1797
767.1795
827.1555828.1354 1133.4736
743
loss of pentose residue743.19 – 611.16 = 132.03
UV spectra of Rutinand Quercetin Trisaccharide
Rutin
+ sugar
Mass Accuracy and Structure Elucidation
High mass accuracy(1 mDa)
Low mass accuracy(100 mDa)
Mass Accuracy and Structure Elucidation
Selected natural compounds with formula C18H19NO (monoisotopic mass 313.1314 Da)
Performing MS-MS: MS-MS of m/z 435 at different collision energies
CE=5 eV
CE=10 eV
CE=15 eV
CE=30 eV
Output Data For High Res. LC-MS Based Metabolomics
AccurateNominal
Software for comparing full-scan datasetsMetAlign method
Peak picking
Alignment
Statistics
Software for comparing full-scan datasets
MarkerLynx method
Out-put from the MarkerLynx (Waters) Program :Analyses of Wild-Type Tomato Peel (Different Stages of Development; ES+)
Principle Components Analysis
Markers Detected
Samples Processed
TIC of currently selected sample
Response of a marker across
all samples
Markers responsible for clustering (by PCA)
A Case Study in Tomato
Small Green
Big Green
Middle Green
Breaker Orange Red
Sample Preparation for Tomato Fruit Metabolome Analysis
Harvesting tomato fruit at different developmental stages
Separation of peel and flesh tissues
Immediate freezing in liquid nitrogen
Grinding to fine powder
Weighing frozen samples
Extraction in 100% MeOH by vortexing and sonication
Analyzing with UPLC-MS [ ES (+) and ES (-) ]
Experimental Set-Up for LC-MS Analysis:PEEL and FLESH during tomato fruit development
Peel
FleshES-
ES+
ES-
ES+Peel
FleshES-
ES+
ES-
ES+
Greenstage
Breakerstage
Peel
FleshES-
ES+
ES-
ES+Peel
FleshES-
ES+
ES-
ES+
Orangestage Red
stage
UPLC-Q-Tof-MS Analysis of Four Stages of Tomato Fruit Development
09-Jan-2006
Time2.00 4.00 6.00 8.00
AU
0.0
5.0e-1
1.0
1.5
2.00 4.00 6.00 8.00
AU
0.0
5.0e-1
1.0
1.5
2.00 4.00 6.00 8.00
AU
0.0
5.0e-1
1.0
1.5
2.00 4.00 6.00 8.00
AU
0.0
5.0e-1
1.0
1.5Tom_24_WTa7 3: Diode Array
Range: 4.3065.980.80
3.25
2.912.262.044.94
3.68 4.135.37
Tom_22_WTa6 3: Diode Array Range: 6.4185.970.80
3.24
2.251.02 2.90 4.943.674.21 5.36
Tom_20_WTa5 3: Diode Array Range: 2.9550.80 5.99
3.25
2.912.261.02
4.953.68 4.13 7.67
Tom_18_WTa4 3: Diode Array Range: 3.20.80
3.26
2.261.02 2.914.95
3.68 7.67
09-Jan-2006
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100Tom_24_WTa7 1: TOF MS ES+
TIC5.00e4
6.010.840.70 1.09
5.171.81 2.403.78 4.23 8.33
Tom_22_WTa6 1: TOF MS ES+ TIC
5.00e4
6.000.85
0.70 5.161.09
2.401.81 3.783.26 4.23 7.71
Tom_20_WTa5 1: TOF MS ES+ TIC
5.00e45.13
5.090.850.70 4.221.09 3.78
2.40 2.595.00 6.02 7.697.39 7.86
Tom_18_WTa4 1: TOF MS ES+ TIC
5.00e45.13
0.840.70 1.09
5.001.82 2.41 3.28 3.78 6.13 7.697.386.51 7.94
UV (240 nm) MS (ES(+),TIC)
RedRed
Orange
Breaker
Green
Orange
Breaker
Green
Out-put from the MarkerLynx (Waters) Program :Analyses of Wild-Type Tomato Peel (Different Stages of Development; ES+)
Peel-Flesh Profiles During Fruit Development
Green peel
Breaker peelBreaker flesh
Green flesh
Red peelOrange peel
Orange flesh
Red flesh
Com
pone
nt 2
Comp. 1
Wild-type Tomato Peel, Different Developmental Stages
09-Jan-2006
Time1.00 2.00 3.00 4.00
%
0
100
1.00 2.00 3.00 4.00
%
0
100
1.00 2.00 3.00 4.00
%
0
100
1.00 2.00 3.00 4.00
%
0
100Tom_24_WTa7 1: TOF MS ES+
3031000Area
3.2742
2.938
Tom_22_WTa6 1: TOF MS ES+ 303
1000Area
3.2740
2.947
Tom_20_WTa5 1: TOF MS ES+ 303
1000Area
3.2836
2.9311
Tom_18_WTa4 1: TOF MS ES+ 303
1000Area
3.2735
2.937
09-Jan-2006
Time2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100Tom_24_WTa7 1: TOF MS ES+
2732.00e4
Area6.01731
Tom_22_WTa6 1: TOF MS ES+ 273
2.00e4Area
6.001158
Tom_20_WTa5 1: TOF MS ES+ 273
2.00e4Area
6.03357
Tom_18_WTa4 1: TOF MS ES+ 273
2.00e4Area
09-Jan-2006
Time2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100Tom_24_WTa7 1: TOF MS ES+
578.41.00e4
Area
5.17484.31
3
3.631
Tom_22_WTa6 1: TOF MS ES+ 578.4
1.00e4Area
5.1690
4.314
Tom_20_WTa5 1: TOF MS ES+ 578.4
1.00e4Area5.13
362
5.078
Tom_18_WTa4 1: TOF MS ES+ 578.4
1.00e4Area
5.12509
5.2410
Red
Orange
Breaker
Green
Tomatine Rutin & Quercetin trisacharide Naringenin chalcone
DOWN NO CHANGE UP
Tomato (var. Ailsa Craig) and the Mutant “Y”,at different ripening stages
Green Breaker Orange Red
Tomato peel
of Mutant “Y” and Wild-type
Wild-Type
Mutant “Y”
Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway
Naringenin chalcone
Cinnamate
Chalcone synthase (CHS)
Anthocyanines
Condensed tannins
Coumarate
Coumaroyl - CoA
Lignins,Coumarins,Chlorogenic-acid
Flavonols
Phenylalanine
Naringenin
Experimental Set-Up for LC-MS Analysis:PEEL and FLESH during “Y” fruit development
Peel
FleshES-
ES+
ES-
ES+Peel
FleshES-
ES+
ES-
ES+
“Y”Breaker
stage
“Y”Greenstage
Peel
FleshES-
ES+
ES-
ES+Peel
FleshES-
ES+
ES-
ES+
“Y”Orange
stage
“Y”Redstage
HPLCHPLC--QTOFQTOF--MS (UltimaMS (Ultima--PRI) PRI) --Wild Type and Wild Type and ““YY”” PeelsPeels
WT-Orange
“Y”-Red
WT-Red
“Y”-Orange
All breaker
All green
1st principal component (X axis) - developmental stage (47.1%) 2nd principal component (Y axis) – mutation (9.4%)
UV and MS Chromatograms of Red Tomato Peel (Wild Type and Mutant “Y”)
09-Jan-2006
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100Tom_56_WTa15 1: TOF MS ES+
TIC2.11e4
0.84
0.70
0.89
1.09
1.815.18
2.403.78
3.642.604.24 4.98
4.86
8.027.95
5.27 5.75
8.31
Tom_8_WTa3 1: TOF MS ES+ TIC
2.12e40.85
0.70
6.021.811.095.16
2.41 3.623.28
2.94
3.77
4.23 4.98
5.43
7.907.63
6.46
8.32
Wild Type
09-Jan-2006
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
AU
0.0
5.0e-1
1.0
1.5
2.0
2.5
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
AU
0.0
5.0e-1
1.0
1.5
2.0
2.5
Tom_56_WTa15 3: Diode Array Range: 2.9160.80
3.25
2.912.041.90 2.20
4.953.68
Tom_8_WTa3 3: Diode Array Range: 3.0220.80
3.25
2.902.332.031.02
5.99
4.943.684.13 5.37
Wild Type
Mutant “Y” Mutant “Y”
UV (240 nm) MS (ES(+),TIC)
IS
The Yellow Pigment Absent in the “Y”Mutant Fruit Peel is Naringenin Chalcone
Tomato peel
s mixture’Standard Naringenin Chalcone Standard
12
13
13 14
15
Tomato peel
12 – Cinnamic acid13 – Naringenin Chalcone14 – Naringenin15 – Kaempferol
m/z100 200 300 400 500 600 700 800 900 1000 1100
%
0
100
%
0
1001: TOF MS ES+
2.35e4273.0797
147.0500
274.0554
275.0623357.0263 583.1375 855.1465629.0711 987.4974 1151.2333
1: TOF MS ES+ 7.90e3273.0789
153.0202
274.0617
357.0297 583.1320 855.1419651.2269 1011.27051060.0111Time5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40
%
0
100
5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40
%
0
100Tom_8_WTa3 1: TOF MS ES+
TIC1.37e4
6.02
5.75
MixAllStand_b1 1: TOF MS ES+ TIC
8.16e36.02
5.93
5.69
6.106.15
6.466.41
Unknown Mass peak (RT=3.51 min) Detected Only in Wild Type
09-Jan-2006
Time2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100Tom_72_WTa19 1: TOF MS ES+
621.1300
7.44 8.39
Tom_56_WTa15 1: TOF MS ES+ 621.1
300
5.755.475.073.080.74 7.69 7.868.40
Tom_40_WTa11 1: TOF MS ES+ 621.1
3003.51
2.35 7.375.07 5.47
Tom_24_WTa7 1: TOF MS ES+ 621.1
3003.51
7.503.64 5.48 8.39
Tom_8_WTa3 1: TOF MS ES+ 621.1
3003.51
2.340.76 7.54
Wild Type
Mutant “Y”
Mutant “Y”
Wild Type
Wild Type
Extracted mass chromatogram at
m/z 621.1 Da
Unknown Mass peak Detected in Higher Levels in “Y”
09-Jan-2006
Time1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15
%
0
100
1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15
%
0
100
1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15
%
0
100
1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15
%
0
100
1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15
%
0
100Tom_72_WTa19 1: TOF MS ES+
265.12.00e3
Area
2.0970
Tom_56_WTa15 1: TOF MS ES+ 265.1
2.00e3Area
2.1067
Tom_40_WTa11 1: TOF MS ES+ 265.1
2.00e3Area
2.1011
Tom_24_WTa7 1: TOF MS ES+ 265.1
2.00e3Area
2.0911
Tom_8_WTa3 1: TOF MS ES+ 265.1
2.00e3Area
2.1010
Wild type
Wild type
Wild-type
Mut. “Y”
Mut. “Y” Extracted mass chromatogram at m/z 265.1 Da
In-depth Analysis of Red Tomato Peel (ES+)Wild-type vs. the “Y” Mutant
MarkerLynxDifferential Markers
- Filtering out non-replicated data- Identifying molecular ions, isotopes, adducts, fragments and passing from Markers to Metabolites- Analysis of differential metabolites and identification
What are the Differentially Expressed Metabolites between WT and “Y” ?
Peak assignment by:- Accurate mass and elemental composition
(MassLynx and natural products database)- MS-MS fragmentation- UV spectrum- Literature - UV & MS spectra databases(e.g. for tomato in PRI)
Differential Metabolites between the "Y" and Wild Type Peel UPLC-QTOF-MS in ES- Mode
Differential metabolites (ESDifferential metabolites (ES--))
"Y" > WT"Y" > WTWT > "Y"WT > "Y"
11111010RR onlyonly
16161212OO onlyonly
442626Both Both RR & & OO
31314848TotalTotal
Differential Metabolites between the "Y" and Wild Type Peel UPLC-QTOF-MS in ES+ Mode
Differential metabolites (ES+)Differential metabolites (ES+)
"Y" > WT"Y" > WTWT >"Y"WT >"Y"
2233RR onlyonly
2200OO onlyonly
881616Both Both RR & & OO
12121919TotalTotal
What are the Differentially Expressed Metabolites between WT and “Y” ?
Most mass peaks showing higher levels in WT than in the “Y” mutant are derivatives of Naringenin Chalcone or Naringenin.
Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway
Naringenin chalcone
Cinnamate
Chalcone synthase (CHS)
Anthocyanines
Condensed tannins
Coumarate
Coumaroyl - CoA
Lignins,Coumarins,Chlorogenic-acid
Flavonols
Phenylalanine
NaringeninIsom
ers
(iden
tical
mas
s)
Hydroxylated Naringenin & Naringenin- Chalcone
Ilana w t-tom rp 23
Tim e27.50 30.00 32.50 35.00 37.50 40.00 42.50
%
0
100
27.50 30.00 32.50 35.00 37.50 40.00 42.50
%
0
100M05983 1: TOF MS ES+
289.07 0.05Da875
26.521270.5037
M05982 1: TOF MS ES+ 289.07 0.05Da
87537.05
289.0717
35.79289.0709
29.95949.2509
28.34580.7736 42.64
273.0382
HPLC-QTOF
“Y”
WT
UV Spectra of Differential Peaks (35.7 min. and 37.05 min.)
nm225 250 275 300 325 350 375 400 425 450 475
AU
0 .0
5.0e-4
1.0e-3
1.5e-3
2.0e-3
AU
0 .0
2.0e-3
4.0e-3
6.0e-3
8.0e-3
1.0e-2
1.126e-2225.6
287.6
M05982 2135 (35.667) Cm (2131:2138-2149:2160) 3: D iode Array 2.195e-3286.6
Peak at 35.7 min
Naringenin
nm250 300 350 400 450 500 550 600
AU
0.0
2.0e-3
4.0e-3A
U
0.0
2.0e-1
4.0e-1
6.0e-16.238e-1366.5740
211.5740
M05982 2209 (36.900) Cm (2209-(2223+2196)) 3: Diode Array 5.444e-3211.5740
376.5740
Naringenin Chalcone
Peak at 37.05 min
Spectra of differential compounds at RT=35.79 min and 37.05 min
RT=37.05 min
RT=35.79 min
Elemental Composition (289.0712)
Elemental composition: C15H12O6 for neutral compound Elemental composition of Naringenin: C15H12O5Difference: OH group instead of H
Suggested Metabolite
MS-MS Fragmentation of m/z 289.07
m/z100 125 150 175 200 225 250 275 300 325
%0
100 274289.0717
163.0419
153.0219
179.0395 271.0564
290.0741
MS-MS fragmentation of m/z 289.0717Eriodictyol (C15H12O6)
Main fragmentations in reference:I. J. M. S. 247 (2005) 93–100
Naringenin and Naringenin ChalconeGlycosides
HPLC-QTOF
“Y”
WT
Ilana Y-tom rp 55
Time28.00 30.00 32.00 34.00
%
0
100
28.00 30.00 32.00 34.00
%
0
100
M05983 1: TOF MS ES+ 435.12 0.10Da
1.41e3
32.79457.2086
28.72929.2811
M05982 1: TOF MS ES+ 435.12 0.10Da
1.41e328.72
435.127430.53
517.1318
29.22565.7620
33.42528.761032.80
457.2038
UV Spectra of Peak at 32.8 min. and Naringenin Chalcone
nm250 300 350 400 450 500 550 600
AU
0.0
1.0e-3
2.0e-3
3.0e-3
AU
0.0
2.0e-1
4.0e-1
6.0e-1
M05982 2544 (42.483) Cm (2544-(2562+2526)) 3: Diode Array 6.774e-1366.6
211.6
M05982 1954 (32.650) Cm (1954-(1965+1944)) 3: Diode Array 3.933e-3366.6211.6
555.6
Peak at 32.8 min
Naringenin Chalcone
MS spectra at RT=28, 30 32 and 33 min
RT=28.7 min
RT=30.5 min
RT=32.8 min
RT=33.4 min
For MS-MS
Exact mass of 435.1273 shows elemental composition of C21H22O10
MS-MS Fragmentation of m/z 435.12 for Peak at RT=32.8
m/z100 150 200 250 300 350 400 450
%
0
100M06007 407 (33.469) Cm (405:411) 2: TOF MSMS 435.13ES+
1.59e3273.0735
153.0214154.0275
435.1260
274.0769
275.0669
436.1255
437.1289
Hexose (m/z 162)
Peak at 32.8 Naringenin Chalcone Naringenin
Chalcone-hexose
Mass Peak at RT=43.40min (>WT)
Selected MassChromatogramm/z 303.08 Da
MS spectra at RT=43.40min TIC chromatogram
Mut MutMut
WT WTWT
Methyl ether of Hydroxylated Naringeninand Naringenin Chalcone
Peaks at 43.4 min and 44.06 min
- Calculated formula is C16H14O6 based on exact mass- Proposed structure for the peak at 43.4 min: Hesperetin or a B-ring positional isomer
MS-MS Fragmentation of m/z 303.08 (43.4 min.)
179153
177
Hesperetin
15.00000000Ilana wt-tom rp 23 msms
m/z100 120 140 160 180 200 220 240 260 280 300 320
%
0
100M06007 505 (43.325) Cm (505:506) 2: TOF MSMS 303.08ES+
369303.0883
153.0225
145.0342
117.0441
177.0581
154.0266179.0368
180.0453 285.0826
304.0926
305.0851
[M+H]+
[M+H-H2O]+
Proposed Scheme of Naringenin & Naringenin Chalcone Hydroxylation, Methylation and Glycosylation
OHOH
OH
OH
O
OH
OOH
OH O
A C
B
Naringenin Chalcone Naringenin
OHOH
OH
OH
O
OH
Hydroxylated Naringenin Chalcone
OHOH
OH O
OHOMe
Methyl ether of Hydroxylated Naringenin Chalcone
OOH
OH O
OHOH
Hydroxylated Naringenine.g. Eriodyctyol or a B-ring isomer
OOH
OH O
OHOMe
Methyl ether of Hydroxylated Naringenine.g.Hesperetin or a B-ring isomer
OOH
OHOH
OH
OH
OO
OH O
Glycosylatione.g. Naringenin glucoside
Glycosylation
Glycosylation
Glycosylation
Glycosylation
Glycosylation
Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway
Naringenin chalcone
Cinnamate
Chalcone synthase (CHS)
Anthocyanines
Condensed tannins
Coumarate
Coumaroyl - CoA
Lignins,Coumarins,Chlorogenic-acid
Flavonols
Phenylalanine
Naringenin