High Resolution LCMS Data Output and Analysis

1
High Resolution LC-MS Data Output and Analysis
2
Mass 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
3
Mass Spectrometer
4. Structural information on metabolite
fragmentation pattern accurate mass
4

• Alternative Ionization Modes

• Atmospheric Pressure Ionization (API), in LC-MS
• Electrospray Ionisation (ESI) polar and
  semi-polar
• Atmospheric Pressure Chemical Ionization (APCI)
  less polar

APCI
ES
water
lipids
polarity of analyte molecule
5
Positive or Negative Modes?
The formation of positive or negative ions
depends on the sign of the applied electrical
field ES (MH) 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)
6
Mass Analyzers

• Ion Cyclotron
• (FT-ICR-MS)

High Resolution Instruments

• Time of Flight
• (TOF)

• Magnetic Sector

• Quadrupole Ion Trap

Low Resolution Instruments

• Quadrupole

7
High Resolution vs. Low Resolution
129
131
130
Low Resolution
High Resolution
8
Mass Analyzers
Mass Accuracy

• Ion Cyclotron
• (FT-ICR-MS)

lt1ppm

• Time of Flight
• (TOF)

3-10ppm

• Magnetic Sector

2-5ppm

• Quadrupole Ion Trap

n/a

• Quadrupole

n/a
9
Mass Accuracy
Caluclated / True / Exact
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 106 ?m
accuracy / mmeasured
?m accuracy
10
Mass Accuracy
High mass accuracy (exact mass measurement)is
usually associated to high resolution
analyzers Unknown compound determination Exact
mass helps to define its atomic composition
11
Resolution
C20H9 C19H7N C13H19N3O2
C20H9
C19H7N
C13H19N3O2
3 different compounds Same nominal mass Low
resolution
3 different compounds 3 different exact
masses High resolution
249
249.0700
249.0580
249.1479
12
Scan Speed (or rate)

• The rate at which we can acquire a mass spectrum,
  (mass units/sec).
• Duel time how much time the portion of the
  sample stays in the detector
• Inter-scan delay time between scans

scan
13
Scan Speed (or rate)

• Will affect the amount of information
  (qualitative and quantitative) that can
  reasonably be attained with a given mass
  analyzer.
• In LC-QTOF-MS 0.2 sec.

14
Your Sample
LC/MS
Result
Sample
Sample
Separation
Efficient
Introduction
Preparation
(column)
Gradient
LC- MS
Today
Data (computer)
Ions
Ions
Ionization
UV
Detection
Separation
MS Interface
Spectra
15
LC-PDA-QTOF-MS of a Tomato Sample
PDA-sample
MS-reference (lock mass) injected every few scans
(TIC)
MS-sample - Total Ion Chromatogram (TIC)
16
Spectra Obtained at Each Scan
PDA-sample
MS-reference (lock mass)
Enkephalin (556.2766 in ES)
MS-sample
17
Extracted chromatogram of a Specific Ion
18
LC/UV/MS Chromatograms of Standards Mixture (UV
at 240nm, ES and ES-)
19
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
20
Chromatogram and Spectrum of Morin
M-H-
ES(-)
ES()
MH
Chromatograms in ES() ES(-) modes
Spectra's in ES() ES(-) modes
21
Adduct Formation in Spectra (Na)
MNa
Rutin C27H30O16
C27H30O16Na
C27H31O16
MH
Na (22)
22
Typical Adducts formed in LCMS
S5
23
Isotopes in Spectra (13C)
Rutin C27H30O16
MNa
C27H30O16Na
13C (little from other isotopes H, O)
C27H31O16
MH
24
Abundance of Isotopes (in Spectra)
25
An example of how isotopes can aid in peak
identification
A 2 (49.3)
A (50.6)
The ratio of peaks containing 79 Br and its
isotope 81 Br (100/98) confirms the presence of
bromine in the compound.
26
Fragments of a Metabolite in Spectra
MS spectrum of Rutin in ES()
MNa
Quercetin
Glucose
MH
Rhamnose
Glucose (162)
Rhamnose (146)
27
Spectra Components

• Molecular Ion
• Fragments of Metabolite
• Adducts (from solvent or di-tri-mers)
• Isotopes

28
Spectrum of Quercetin Trisaccharide (a Rutin
Derivative)
sugar
29
Mass Accuracy and Structure Elucidation
High mass accuracy (1 mDa)
Low mass accuracy (100 mDa)
30
Mass Accuracy and Structure Elucidation
Selected natural compounds with formula C18H19NO
(monoisotopic mass 313.1314 Da)
31
Performing MS-MS MS-MS of m/z 435 at different
collision energies
32
Output Data For High Res. LC-MS Based Metabolomics
Accurate
Nominal
33
Software for comparing full-scan datasets
MetAlign method
Peak picking
Alignment
Statistics
34
Software for comparing full-scan datasets
MarkerLynx method
35
Out-put from the MarkerLynx (Waters) Program
Analyses of Wild-Type Tomato Peel (Different
Stages of Development ES)
Samples Processed
Markers Detected
Response of a marker across all samples
TIC of currently selected sample
Markers responsible for clustering (by PCA)
Principle Components Analysis
36
A Case Study in Tomato
Small Green
Breaker
Orange
Red
Big Green
Middle Green
37
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 (-)
38
Experimental Set-Up for LC-MS Analysis PEEL and
FLESH during tomato fruit development
Green stage
Breaker stage
Orange stage
Red stage
39
UPLC-Q-Tof-MS Analysis of Four Stages of Tomato
Fruit Development
Red
Red
Orange
Orange
Breaker
Breaker
Green
Green
UV (240 nm)
MS (ES(),TIC)
40
Out-put from the MarkerLynx (Waters) Program
Analyses of Wild-Type Tomato Peel (Different
Stages of Development ES)
41
Peel-Flesh Profiles During Fruit Development
Orange peel
Red peel
Red flesh
Orange flesh
Component 2
Breaker flesh
Breaker peel
Green peel
Green flesh
Comp. 1
42
Wild-type Tomato Peel, Different Developmental
Stages
UP
DOWN
NO CHANGE
43
Tomato (var. Ailsa Craig) and the Mutant Y, at
different ripening stages
Wild-Type
Mutant Y
Tomato peel of Mutant Y and Wild-type
44
Biosynthesis of Naringenin Chalcone in the
Flavonoid Pathway
Phenylalanine
Cinnamate
Lignins, Coumarins, Chlorogenic-acid
Coumarate
Coumaroyl - CoA
Chalcone synthase (CHS)
Naringenin chalcone
Anthocyanines
Flavonols
Naringenin
Condensed tannins
45
Experimental Set-Up for LC-MS Analysis PEEL and
FLESH during Y fruit development
Y Green stage
Y Breaker stage
Y Orange stage
Y Red stage
46
UV and MS Chromatograms of Red Tomato Peel (Wild
Type and Mutant Y)
47
The Yellow Pigment Absent in the Y Mutant Fruit
Peel is Naringenin Chalcone
13
Tomato peel
Tomato peel
273.0789
14
13
6.02
6.10
6.15
15
12
5.93
6.46
6.41
5.69
Naringenin Chalcone Standard
Standards mixture
274.0617
651.2269
357.0297
153.0202
583.1320
855.1419
1011.2705
1060.0111
12 Cinnamic acid 13 Naringenin Chalcone 14
Naringenin 15 Kaempferol
48
Unknown Mass peak (RT3.51 min) Detected Only in
Wild Type
Extracted mass chromatogram at m/z 621.1 Da
49
Unknown Mass peak Detected in Higher Levels in Y
Extracted mass chromatogram at m/z 265.1 Da
Mut. Y
Mut. Y
Wild-type
Wild type
Wild type
50
In-depth Analysis of Red Tomato Peel
(ES) Wild-type vs. the Y Mutant
MarkerLynx Differential Markers
Filtering in MS Excel Matlab and subsequently
peak analysis by eye
51
HPLC-QTOF-MS (Ultima-PRI) - Wild Type and Y
Peels
1st principal component (X axis) - developmental
stage (47.1) 2nd principal component (Y axis)
mutation (9.4)
All breaker
WT-Orange
WT-Red
All green
Y-Orange
Y-Red
52
Post Metalignall WT gt Y mass peaks
RT in umin
M/z
Scan
53
HPLC-qTOF -MarkerLynx
HPLC-qTOF-MetAlign
HPLC-qTOF-XCMS
54
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)

55
Differential Metabolites between the "Y" and Wild
Type Peel UPLC-QTOF-MS in ES- Mode
56
Differential Metabolites between the "Y" and Wild
Type Peel UPLC-QTOF-MS in ES Mode
57
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.
58
Biosynthesis of Naringenin Chalcone in the
Flavonoid Pathway
Phenylalanine
Cinnamate
Lignins, Coumarins, Chlorogenic-acid
Coumarate
Coumaroyl - CoA
Chalcone synthase (CHS)
Naringenin chalcone
Anthocyanines
Flavonols
Naringenin
Condensed tannins
59
Hydroxylated Naringenin Naringenin- Chalcone
HPLC-QTOF
Y
WT
60
UV Spectra of Differential Peaks (35.7 min. and
37.05 min.)
Naringenin Chalcone
Naringenin
Peak at 37.05 min
Peak at 35.7 min
61
Spectra of differential compounds at RT35.79 min
and 37.05 min
RT35.79 min
RT37.05 min
62
Elemental Composition (289.0712)
Elemental composition C15H12O6 for neutral
compound Elemental composition of Naringenin
C15H12O5 Difference OH group instead of H
63
Suggested Metabolite
MS-MS Fragmentation of m/z 289.07
MS-MS fragmentation of m/z 289.0717
Eriodictyol (C15H12O6) Main fragmentations in
reference I. J. M. S. 247 (2005) 93100
64
Naringenin and Naringenin Chalcone Glycosides
65
MS spectra at RT28, 30 32 and 33 min
Exact mass of 435.1273 shows elemental
composition of C21H22O10 m/z 435.1273 is an
hexose derivative of Naringenin or NarChalc
For MS-MS
66
MS-MS Fragmentation of m/z 435.12 for Peak at
RT32.8
Peak at 32.8
Naringenin Chalcone
Naringenin Chalcone-hexose
Hexose (m/z 162)
67
Selected Mass Chromatogram m/z 303.08 Da
MS spectra at RT43.40min
TIC chromatogram
Mut
Mut
Mut
WT
WT
WT
68
UV Spectra of Peak at 32.8 min. and Naringenin
Chalcone
Peak at 32.8 min
Naringenin Chalcone
69
Methyl ether of Hydroxylated Naringenin and
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 Proposed
structure for the peak at 44.06 min Naringenin
Chalcone isomer
70
MS-MS Fragmentation of m/z 303.08 (43.4 min.)
Hesperetin
71
Proposed Scheme of Naringenin Naringenin
Chalcone Hydroxylation, Methylation and
Glycosylation
Glycosylation
Naringenin Chalcone
Naringenin
Glycosylation e.g. Naringenin glucoside
Glycosylation
Glycosylation
Hydroxylated Naringenin Chalcone
Hydroxylated Naringenin e.g. Eriodyctyol or a
B-ring isomer
Glycosylation
Glycosylation
Methyl ether of Hydroxylated Naringenin Chalcone
Methyl ether of Hydroxylated Naringenin e.g.Hesper
etin or a B-ring isomer
72
Biosynthesis of Naringenin Chalcone in the
Flavonoid Pathway
Phenylalanine
Cinnamate
Lignins, Coumarins, Chlorogenic-acid
Coumarate
Coumaroyl - CoA
Chalcone synthase (CHS)
Naringenin chalcone
Anthocyanines
Flavonols
Naringenin
Condensed tannins