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What Are You Missing in Your Metabolome…The Orbitrap Perspective
2
Agenda
Introduction to metabolomics
Addressing the challenges with high resolution accurate mass
spectrometry
Experimental design with Thermo Scientific™ Orbitrap™ mass
spectrometers
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3
1
3
genome transcriptome proteome metabolome
Metabolomics – the Link between Genotype and Phenotype
environment
phenotype
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Human Plasma Metabolomics and Exercise
Why is exercise good for health?
Lewis, G.D., Gerszten, R.E. et al (2010) Metabolic Signatures of Exercise in Human Plasma
Science Translational Medicine 2(33): 33ra37.
↑ niacinamide
↑ glycerol
↑ glucose-6-phosphate
↑ pantothenate
6
Untargeted Metabolomics: Challenges
Structural and physical diversity
Isomeric / isobaric species
Dynamic range Large sample sets
leucine isoleucine
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High Quality Data for High Quality Results
• Complex matrix
• Differentiate similar masses
• Isobaric species
• Find isotopic pattern
High Resolution
• Identification of unknowns
• Narrow mass tolerance
• Mass stability from peak to peak and run to run
Mass Accuracy
• Scan-to-scan consistency
• Injection-to-injection reproducibility
• Robustness over extended time periods
Instrument Performance
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Untargeted Metabolomics Workflow
Collect and prepare samples
Untargeted discovery Compound Detection,
Quantitation, and
Identification
IC-MS
LC-MS
GC-MS
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Thermo Scientific™ Q Exactive™ Series Portfolio for Metabolomics
VALUE
PE
RF
OR
MA
NC
E
Thermo Scientific™ Q Exactive™ Focus MS• Orbitrap analyzer
• Mass Range m/z 50 - 3000
• Mass Accuracy <1ppm
• Max. Mass Resolution >70,000
• Scan speed up to 12Hz
• Spectral multiplexing
• Polarity switching <1 sec
Thermo Scientific™ Q Exactive™ MS
• Orbitrap analyzer
• Mass Range m/z 50 - 6000
• Mass Accuracy <1ppm
• Max. Mass Resolution >140,000
• Scan speed up to 12Hz
• Spectral Multiplexing
• Polarity switching <1 sec
Thermo Scientific™ Q Exactive™ Plus MS
• Orbitrap analyzer• Mass Range m/z 50 - 6000
• Mass Accuracy <1ppm• Max. Mass Resolution >140,000• Scan speed up to 12Hz
• Spectral multiplexing
• Polarity switching <1 sec
• Advanced Quadrupole Technology (AQT)
• Advanced Active Beam Guide (AABG)
• Opt. Enh Res.Mode (280k)
Thermo Scientific™ Q Exactive™ HF MS
• Ultra High Field Orbitrap analyzer
• Mass Range m/z 50 - 6000
• Mass Accuracy <1ppm
• Max. Mass Resolution >240,000
• Scan speed up to 18Hz
• Advanced Quadrupole Technology (AQT)
• Advanced Active Beam Guide (AABG)
• Spectral Multiplexing
• Polarity switching <1 sec
Thermo Scientific™ Q Exactive ™ HF-X MS• Ultra High Field Orbitrap analyzer
• Mass Range m/z 50 - 6000
• Mass Accuracy <1ppm
• Max. Mass Resolution >240,000
• Scan speed up to 40Hz
• High capacity transfer tube
• Electrodynamic ion funnel
• Advanced QuadrupoleTechnology (AQT)
• Advanced Active Beam Guide (AABG)
• Spectral Multiplexing
• Polarity switching <1 sec
• Advanced ddHCD algorithm
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Ion Chromatography (IC): Excellent Separation Tool for Polar Metabolites
Thermo Scientific™ Dionex™ ICS-5000+ HPIC System
Thermo Scientific™ Q Exactive™ Orbitrap Series MS
RT: 4.11 - 16.79 SM: 7G
5 6 7 8 9 10 11 12 13 14 15 16
0
2
4
6
8
10
12
14
16
18
20
22
24
0
2
4
6
8
10
12
14
16
18
20
22
24
NL:1.82E7
m/z= 259.0196-259.0248 MS 50ppb_isotopes_512_UM1E-2
NL:1.37E7
m/z= 338.9854-338.9922 MS 50ppb_isotopes_512_UM1E-2
9 Diphosphate sugar
isomers
20
0
10
11 Mono-phosphate sugar
isomers
RT: 4.11 - 16.79 SM: 7G
5 6 7 8 9 10 11 12 13 14 15 16
0
2
4
6
8
10
12
14
16
18
20
22
24
0
2
4
6
8
10
12
14
16
18
20
22
24
NL:1.82E7
m/z= 259.0196-259.0248 MS 50ppb_isotopes_512_UM1E-2
NL:1.37E7
m/z= 338.9854-338.9922 MS 50ppb_isotopes_512_UM1E-2
20
0
10
#9, F6P
#10, F6P
• Efficient and sensitive separation for small
isobaric polar metabolites
• Increased sensitivity, > 10-fold, as result of
low chemical noise
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Q Exactive GC: A New Chapter in GC-MS Based Metabolomics
Offering Unprecedented Depth in GC-MS Analysis
• Resolution up to 100K
• Sub-ppm mass accuracy
• > 6 orders dynamic range
• Triple quad-like sensitivity
Thermo Scientific™ Q Exactive™ GC
Hybrid Quadrupole-Orbitrap GC-MS/MS System
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Vanquish UHPLC Systems Bring You
To improve separations
• Binary and Quaternary pumping systems
• Widest flow-pressure footprint
• Optimized system fluidics
Confidence Performance
AccuracySensitivity
To control experiments
• Unsurpassed sample dosage
• Active and passive pre-heating
• Multiple thermostatting options
To ensure highest data quality
• Unmatched retention time precision with SmartInject
• Automation of workflows with barcode reading
• Predictive performance indicators
To find the complete picture
• UV detection optimized for lowest dispersion and minimal baseline noise
• Highly sensitive and selective fluorescence detection
• Near universal charged aerosol detection
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Commercially Available Reference Standard
• Human plasma sample with certified values for most common metabolites.
• Organic solvent precipitation of proteins and extraction of small molecules.
http://srm1950.nist.gov/
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Untargeted Metabolomics Workflow
http://srm1950.nist.gov/
Case Study: NIST SRM 1950 – a commercially available reference standard
Unbiased detection and
confident identification
tools
Thermo Scientific™ Q Exactive™ HF mass spectrometer
Thermo Scientific™ Vanquish™ UHPLC system
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Presenter:
JAKOBMass Accuracy Provides Selectivity
Example: Folic acid (C19H19N7O6)
has a protonated ion at 442.1470
Mass Error (tolerance)
# of Possible Chemical
Formulae(using only C, H, N & O)
0.1 Da 319
10 ppm 14
5 ppm 6
2 ppm 2
Orbitrap HRAM MS delivers confident identification of unknowns
Mass error (ppm) = exact mass – accurate mass/MW*106
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Stable Mass Accuracy from Scan to Scan Across the Peak
creatine
theoretical m/z 132.0768
Orbitrap MS provides confidence in peak detection
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Excellent Mass Accuracy Across the Molecular Weight Range
Orbitrap MS delivers accurate mass measurements for all metabolites
thyroxinecreatine
glycine
Metabolites identified from human plasma and verified against an authentic standard
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Excellent Mass Accuracy Across the Molecular Weight Range
Orbitrap MS delivers accurate mass measurements for all metabolites
Metabolites identified from human plasma and verified against an authentic standard
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Excellent Mass Accuracy During Fragmentation
60 80 100 120 140 160 180 200
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abu
nd
an
ce
162.05486C9 H8 O2 N
-0.62 ppm
190.04973C10 H8 O3 N
-0.74 ppm
116.04940C8 H6 N
-0.65 ppm89.03854
C 7 H5
-0.37 ppm 134.06003C8 H8 O N
-0.09 ppm
kynurenic acid
MS/MS of m/z 190
Orbitrap MS provides high mass accuracy for confident structure annotation
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Robust Mass Accuracy from Run to Run
Orbitrap MS delivers unmatched mass accuracy over long periods of time
Human plasma (NIST SRM1950)
spiked with internal standards
Mass accuracy <2ppm in both
positive and negative modes
during a 55 h analysis
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Orbitrap MS: Unmatched Resolution
0
50000
100000
150000
200000
250000
300000
350000
100 200 300 400 500 600 700 800 900 1000
Resolu
tion (
FW
HM
)
Orbitrap MS: Q Exactive HF QTOF Competitor S
QTOF Competitor A QTOF Competitor B
m/z
Superior resolution ideal for
small molecule analysis
High Orbitrap MS resolving power for correct identification of isomeric and isobaric species
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Resolving Isobaric Metabolites in Human Plasms with Orbitrap High Resolution
175.09 175.10 175.11 175.12 175.13m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive A
bundance
arginine
m/z 175.11888
N-acetyl ornithine
m/z 175.10764
65ppm
15K
30K
60K
120K
240K
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High Resolution is Essential for Fine Isotopic Pattern Determination
150.0 150.5 151.0 151.5 152.0 152.5m/z
0
50
100
Rela
tive A
bun
dan
ce
150.0582
151.0615 152.0539
151.04 151.05 151.06 151.07m/z
0
50
100
Rela
tive A
bundance
15K
30K
60K
120K
240K
L-Methionine C5H11NO2S
Positive mode
-0.7ppm
A0
A1 A2
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High Resolution is Essential for Fine Isotopic Pattern Determination
150.0 150.5 151.0 151.5 152.0 152.5m/z
0
50
100
Rela
tive A
bun
dan
ce
150.0582
151.0615 152.0539
151.04 151.05 151.06 151.07m/z
0
50
100
Rela
tive A
bundance
120K
L-Methionine C5H11NO2S
Positive mode
-0.7ppm
A0
A1 A2
13C
2H
33S15N
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Mass Resolution and Scan Speed
R = 30k
R = 60k
R = 120k
~4.8 s
Scans/peak = 67
Scan speed = 14 Hz
Scans/peak = 36
Scan speed = 7.5 Hz
Scans/peak = 19
Scan speed = 4 Hz
Vanquish UHPLC and Q Exactive HF MS
D8-Phenylalanine spiked in human plasma
~4.8 s
~4.8 s
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High Resolving Power Increases Metabolome Coverage
1106
15841719
0
500
1000
1500
2000
30K 60K 120K
co
mp
ou
nd
s d
ete
cte
d
resolution
43% increase
9% increase
Human plasma metabolites
(positive mode)
Human plasma metabolites
(negative mode)
resolution
548
799900
0
200
400
600
800
1000
30K 60K 120K
co
mp
ou
nd
s d
ete
cte
d
46% increase
13% increase
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Improved Metabolome Coverage with the Q Exactive HF MS
R = 70k
QE Plus
Scans/peak = 18
Scan speed = 3.8 Hz
~4.8 s
D8-Phenylalanine spiked in human plasma
Ultra-High Field
Orbitrap
Q Exactive HF MS
Standard Orbitrap
Q Exactive Plus MS
R = 60k
QE HF
Scans/peak = 36
Scan speed = 7.5 Hz
~4.8 s
30
Improved Metabolome Coverage with the Q Exactive HF MS
R = 70k
QE Plus
Scans/peak = 18
Scan speed = 3.8 Hz
~4.8 s
D8-Phenylalanine spiked in human plasma
Ultra-High Field
Orbitrap
Q Exactive HF MS
Standard Orbitrap
Q Exactive Plus MS
R = 120k
QE HF
Scans/peak = 19
Scan speed = 4 Hz
~4.8 s
31
Improved Metabolome Coverage with the Q Exactive HF MS
1162
15841719
0
500
1000
1500
2000
QE Plus -70K
QE HF -60K
QE HF -120K
co
mp
ou
nd
s d
ete
cte
d
36% increase
9% increase
Human plasma metabolites
(positive mode)
R = 70k
QE Plus
Scans/peak = 18
Scan speed = 3.8 Hz
~4.8 s
D8-Phenylalanine spiked in human plasma
R = 120k
QE HF
Scans/peak = 19
Scan speed = 4 Hz
~4.8 s
35
Maximize Unknown Compound Detection with MS1 Only Acquisition
Full MS
R = 120k
~5.4 s
Scans/peak = 23
Scan speed = 4 Hz
D8-Valine spiked in human plasma
Full MS
Top5 ddMS2
R = 120k
~5.4 s
Scans/peak = 9
Scan speed = 1.7 Hz
Full MS
R = 120k
~3.6 s
Scans/peak = 16
Scan speed = 4 Hz
Full MS
Top5 ddMS2
R = 120k
Scans/peak = 6
Scan speed = 1.7 Hz
~3.6 s
Endogenous palmitoylcarnitine in human plasma
36
Maximize Unknown Compound Detection with MS1 Only Acquisition
Full MS
R = 120k
~5.4 s
Scans/peak = 23
Scan speed = 4 Hz
D8-Valine spiked in human plasma
Full MS
Top5 ddMS2
R = 120k
~5.4 s
Scans/peak = 9
Scan speed = 1.7 Hz
acquisition mode
17191883
0
500
1000
1500
2000
Full MS -ddMS2
MS1 only
co
mp
ou
nd
s d
ete
cte
d
10% increase
Human plasma metabolites
(positive mode)
37
High Quality Data Ensure High Quality Results
Instrument robustness
High resolution
Mass accuracy
unknown identification
high quality data
enhanced experimental
design