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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

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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

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Untargeted Metabolomics: A Universal Application

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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





• Spectral Multiplexing


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


• 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





• Advanced Quadrupole Technology (AQT)




Thermo Scientific™ Q Exactive ™ HF-X MS• Ultra High Field Orbitrap analyzer





• High capacity transfer tube

• Electrodynamic ion funnel

• Advanced QuadrupoleTechnology (AQT)




• 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

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24

0

2

4

6

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NL:1.82E7

m/z= 259.0196-259.0248 MS 50ppb_isotopes_512_UM1E-2

NL:1.37E7


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

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0

2

4

6

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10

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14

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22

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NL:1.82E7


NL:1.37E7


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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High Quality Data:

Accurate Mass Measurements

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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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High Quality Data:

High Mass Resolution

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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

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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)


(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


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


R = 70k

QE Plus

Scans/peak = 18

Scan speed = 3.8 Hz

~4.8 s


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

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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


(positive mode)

R = 70k

QE Plus

Scans/peak = 18

Scan speed = 3.8 Hz

~4.8 s


R = 120k

QE HF

Scans/peak = 19

Scan speed = 4 Hz

~4.8 s

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High Quality Data:

Experimental Design for Discovery Metabolomics

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Unknown Discovery Metabolomics Workflow

Disease Matched Controls

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Discovery Metabolomics Workflow

Pooled

sample

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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

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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


(positive mode)

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High Quality Data Ensure High Quality Results

Instrument robustness

High resolution

Mass accuracy

unknown identification

high quality data

enhanced experimental

design

38

Questions?

what are you missing in your metabolome the orbitrap ... quality data for high quality results ......

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