new mass spectrometry tools to transform metabolomics and ... · 2 omics & the central dogma of...

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July.3.2013 Ken Miller Vice President of Marketing,

Life Sciences Mass Spectrometry

New Mass Spectrometry Tools to Transform Metabolomics and Lipidomics

2

Omics & the Central Dogma of Biology

DNA

RNA

Proteins

Metabolites Lipids

What has occurred

Genomics – 22,000 genes Transcriptomics – 100,000 transcripts Proteomics – 1,000,000 proteoforms

Metabolomics – 2,000 compounds Lipidomics – 10,000 species

What could occur

What is occurring

What may occur

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Addressing the Metabolomics Challenges

Developing SOPs for major sample types

Innovating LC/MS to improve separation & detection

Developing streamlined, workflow-oriented software

Building and connecting to new and existing database

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Orbitrap Delivers the Highest Quality Data

• Highest Resolution

• Best Mass Accuracy

• Widest Dynamic Range

• Excellent Sensitivity

Better data, Better results!

5

LCMS Metabolomics Offerings

Profile

• Detect, align, and quantify components, use statistical analysis to find key features

Identify

• Putative ID web or local DB search • Confirm ID via MS2

• De novo structural ID via MSn

Quantify

• Rountinely quantify sets of putative biomarkers in large sample populations

Quan / Qual analysis of endogenous molecules of low molecular mass (<1200-1500Da)

High-End Budget Limited

Thermo Scientific™ Q Exactive™ MS

Thermo Scientific™ Exactive™ Series MS

Thermo Scientific™ Orbitrap Fusion™ MS

TSQs

Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC

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Orbitrap Fusion

Unmatched Analytical Performance

Revolutionary performance

Unprecedented usability

Exceptional versatility

Orbitrap Fusion Tribrid Mass Spectrometer

Tribrid: (trī-brǝd), n. three Mass Analyzers working in parallel to produce unmatched analytical results

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2

3

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Active beam guide (ABG)

Ultra-high-field Orbitrap mass analyzer

Dual-pressure linear ion trap

Ion-routing multipole

Quadrupole mass filter

EASY-ETD (and IC) ion source


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Flexible, Intuitive, Easy to Learn Software

The Thermo Scientific™ Orbitrap Fusion™ Method Editor allows for drag and drop method building.

Dynamic Scan Management for maximum data quality

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Orbitrap Fusion Tribrid MS

Unmatched Analytical Performance 450,000 Resolution to remove spectral interferences

CID/HCD/ETD detected by the Ion Trap or Orbitrap at any level of MSn for maximum experimental flexibility

Powered by the new Dynamic Scan Management architecture that lets every user easily get the maximum information from every sample

Scan rate FTMS2 15 Hz

Scan rate ITMS2 20 Hz

Max resolution 450K at m/z 200

Quad isolation down to 0.4 amu

Ion trap isolation down to 0.2 amu

Mass Accuracy 1 ppm

Dissociation CID, HCD, ETD

MSn Up to MS10 in ion trap or Orbitrap

Analyzers Q, FTMS, ITMS

Detectors Ion Trap, Orbitrap

Compact 1186 x 674 x 650 mm (w, d, h)

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Mass Error (ppm)

# of Possible Chemical Formulae (using only C, H, N & O)

165 209 10 13 5 7 3 4 2 2 1 2

0.5 1 (0.0003 daltons)

Mass Accuracy Matters!

ppm error = (mass error / MW) x 106

Accurate mass improves the ability to confidently identify unknown compounds

EXAMPLE: • Reserpine (C33H40N2O9) has a protonated ion at 609.28066

(0.1 daltons)

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Challenge Orbitrap Fusion MS Generate absolutely most accurate data to facilitate compound identification.

EASY IC (internal calibration): new ETD source allows use of internal calibration for sub-ppm mass accuracies. On/off toggle in SW. No ion suppression by IC.

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Reagent/Internal Calibrant Source

Tube lens is replaced with discharge ion source

for IC & ETD

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Internal Calibration: LC/MS of Omeprazole Metabolites

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1 2 3 4 5 6 7 8 9 0

100

R e l

a t i v

e I n

t e n s

i t y

Time (min)

362.11690 m/z

N

N H O

O S N

O

= O

H+


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3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Time (min)

0.55 ppm

0.19 ppm

0.36 ppm

0.28 ppm

0.47 ppm

0.35 ppm

0.55 ppm

0.28 ppm

0.28 ppm

0.03 ppm

-0.06 ppm

0.11 ppm 0.19 ppm

0.02 ppm

0.47 ppm

0.55 ppm 0.55 ppm

0.55 ppm

0.47 ppm

0.55 ppm

0.19 ppm

0.19 ppm

-0.14 ppm 0.19 ppm


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3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Time (min)

0.55 ppm

0.19 ppm

0.36 ppm

0.28 ppm

0.47 ppm

0.35 ppm

0.55 ppm

0.28 ppm

0.28 ppm

0.03 ppm

-0.06 ppm

0.11 ppm 0.19 ppm

0.02 ppm

0.47 ppm

0.55 ppm 0.55 ppm

0.55 ppm

0.47 ppm

0.55 ppm

0.19 ppm

0.19 ppm

-0.14 ppm 0.19 ppm

3.70 3.75 3.80 Time (min)

-0.80 ppm

-0.55 ppm 0.03 ppm

0.47 ppm

-0.22 ppm 0.03 ppm

0.19 ppm 0.97 ppm

-0.14 ppm

0.11 ppm

-0.05 ppm

0.03 ppm 0.28 ppm

0.11 ppm

0.28 ppm


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Easy-IC Providing Sub-ppm Mass Accuracy in MS and MS/MS

2.76 2.77 2.78 2.79 2.80 2.81 2.82 2.83 2.84 2.85 2.86Time (min)

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70

80

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1000

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205.09721

205.09724

205.09721

205.09723

205.09717

205.09717205.09717205.09715 205.09718 205.09720

205.09749

205.09747

205.09741205.09746

205.09740205.09770

205.09772205.09746 205.09735 205.09738

80 100 120 140 160 180 200m/z

0

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146.06033

118.06544

132.08105 188.07079170.06030

MS HCD MS/MS

80 100 120 140 160 180 2000

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Rel

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146.06020

118.06530

132.08090 170.06015 188.0706391.05422

∆ 0.2 ppm O

NH

OH

NH

NH

OHO

NH

∆ 0.9 ppm

∆ 1.4 ppm

∆ 0.7 ppm NH

•

∆ 0.4 ppm

∆ 0.8 ppm

N2H

∆ 1.4 ppm

NH

N 2HNH

∆ -0.1 ppm

∆ 1.1 ppm

∆ 1.8 ppm

∆ 2.1 ppm

∆ 2.0 ppm

∆ 1.7 ppm

∆ 1.9ppm

∆ 2.9 ppm

∆ 1.1 ppm

Orbitrap Fusion MS operating @120K for FS, 15k for HCD MS2, with UHPLC. Sample, Urine. Analyte: L-Tryptophan, C11H12N2O2, [M+H]+=205.09715

With IC Avg ∆= 0.4 ppm

With IC

Without IC Without IC Avg ∆= 1.5 ppm

ASMS 2013 poster MP045 by Junhua Wang et al.

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Challenge Orbitrap Fusion MS Separation of distinct isobars in complex mixtures

Orbitrap Fusion can achieve up to 450,000 resolution.

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151.05 151.06m/z

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R=120k

151.05 151.06m/z

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R=60k

151.05 151.06m/z

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R=240k

151.05 151.06m/z

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R=30k

See the Fine Isotope Pattern with Ultra-high Resolution

L-Methionine C5H11NO2S PositiveMode20130518092831 #656 RT: 2.64 AV: 1 NL: 1.48E8T: FTMS + p ESI Full ms [100.00-212.08]

150.0 150.5 151.0 151.5 152.0m/z

0

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150.0584R=627006

151.0618R=621502

152.0542R=601802

151.05 151.06m/z

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R=450k

0.7ppm Positive Observed

C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:40) Chrg 1 ...

150.0 150.5 151.0 151.5 152.0m/z

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150.0583R=499553

151.0617R=499368

152.0541R=499443

Positive Simulated R=450,000

0.0ppm

C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:40) Chrg 1 ...

151.050 151.055 151.060 151.065m/z

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151.0617 C4

13CH12O2NS

151.0625 C5H12O17ONS 151.0646 C5

13C14CH918O17O

151.0577 C5H12O2N33S

151.554 14C4H10

18O233S


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160 165 170 175 180 185 190 195 200 205 210 215 2200

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215.03278R=356104167.02103

R=353605

187.00713R=333105172.99141

R=497902 180.06662R=561707 210.84235

R=535102203.02049R=513702

Resolving the Most Complex Sample Nano-infusion of NIST SRM1950 plasma extract, Negative mode

Paraxanthine C7H8N4O2

D-Glucose C6H12O6

D-Glucose or Paraxanthine ?

179.050 179.055 179.060 179.0650

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179.0573R=67606

179.050 179.055 179.060 179.0650

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179.0574R=148204

179.0558R=130000

179.050 179.055 179.060 179.0650

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100R

elat

ive

Abu

ndan

ce

179.0574R=304604

179.0560R=275000

179.050 179.055 179.060 179.0650

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179.0574R=607806

179.0560R=562802

R=60,000 R=120,000

R=240,000 R=450,000

1.1 ppm

0.55 ppm

0.55 ppm

0.55 ppm

1.7 ppm

0.56 ppm

0.55 ppm

Confirmed with STD mixture.

179.055 179.060m/z

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179.05763R=147660

179.055 179.060m/z

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179.05759R=280705

179.055 179.060m/z

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179.05747R=72040

179.055 179.060m/z

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179.05743R=516802

179.05609R=537700

R=60,000 R=120,000

R=240,000 R=450,000

D-Glucose

Paraxanthine

0.55 ppm

0.55 ppm

179.0561 or 179.0575?


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Challenge Orbitrap Fusion MS Achieve maximum coverage of the metabolome/lipidome. Many molecules require special fragmentation conditions to reveal their structural information.

Instrument architecture allows for extremely fast MS/MS and full flexibility of MSn experiments for structural analysis including novel workflows such as HCD3 and MS2 HCD followed by MS3 CID.

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RT: 1.6345 - 1.8627 SM: 5G

1.64 1.66 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1Time (min)

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1.7969

1.7879

1.8059

1.7789

1.8149

1.6980 1.7699

1.7160 1.82411.6798 1.76101.72511.6707 1.7520 1.83331.6525 1.85161.7298

1.8393

1.6572 1.71181.6846 1.7401 1.7927 1.81071.76571.6677

MS in Orbitrap ↓

Data dependent HCD MS/MS in Orbitrap

Full MS MS OT, R=70K

MS2 Top1

HCD MS2 OT, R=17.5K MS2 Top2

Full MS MS OT, R=60K

MS2 Top1 MS2 Top2 MS2 Top3 MS2 Top4 MS2 Top5

0.5s

Method 1: The “Super” Q Exactive MS Mode

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RT: 2.5298 - 2.5478

2.530 2.535 2.540 2.545Time (min)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Rel

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2.5384 2.5465

2.53942.5315 2.54322.5350

2.5371 2.5445

NL:1.68E8Base Peak F: ms MS E3_FS60k_Urine_HCDIT_superIT500ms

NL:3.44E6Base Peak F: ms2 MS E3_FS60k_Urine_HCDIT_superIT500ms

Full MS MS Orbitrap, R=240K

MS2 Top1 MS2 Top2 MS2 Top3 MS2 Top4 MS2 Top5

0.5s

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Method 3: MSn Enables de novo Compound ID

Standards HR/AM MSn Library

Unknowns

Standards Standards IDs

Match Score Above Threshold

Individual Spectrum Search

Partial Structures

Structure Candidates Tr

ee S

earc

h

Infusion (or LC)

LC-MSn

Add

Match Score Below Threshold

• MSn technology • Spectral library technology • Algorithms for structural-

spectral investigation

Sheldon, Mistrik, Croley, J Am Soc Mass Spectrom, 2009, 20: 370-376

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Method 3: HR/AM Ion Trees for Unknown Identification

HCD/CID MS2 in IT/OT

HCD/CID MS3 in IT/OT

3.178 3.180 3.182 3.184 3.186 3.188 3.190Time (min)

0

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Rel

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10

20

3.1791 3.1891

MS

HCD MS2 IT

0.6 sec

CID MS3 OT

MS2 Top1 MS2 Top2

MS2 Top1 MS2 Top2 MS2 Top3 MS2 Top4

Full MS in Orbitrap ↓

Data dependent HCD MS2 in ion trap

↓ Data dependent CID MS3

in Orbitrap

De novo structural elucidation: capture substructure with MSn ion tree

Full MS

0.5-0.8s

26

Ion Routing Multipole

C-Trap

Ion Trap

HPC

LPC

Orbitrap

MP0 Q1

Full Orbitrap MS scan

Parent ion isolation in Q1 and HCD MS2> OTMS detection of MS2 fragments HCD fragment isolation in the ion trap and HCD MS3

Orbitrap MS detection of HCD MS3 fragments

OTMS> HCD OTMS2> HCD OTMS3

m/z

Full FTMS Scan

m/z

HCD FTMS2 Scan

m/z

HCD FTMS3 Scan

HCD MSn is Unique to Orbitrap Fusion MS

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60 80 100 120m/z

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Rel

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96.0556

69.0447

60 80 100 120 140 160 180 m/z

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Rel

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unda

nce

124.02

105.01

180.96 141.91

95.08 69.01

Structural Homology Identified by MSn Ion Tree Search

3.178 3.180 3.182 3.184 3.186 3.188 3.190Time (min)

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20NL: 3.38E6Base Peak m/z= 181.0703-181.0739 MS b667JWUrine_HCDIT_CIDOTtop2_500ms

NL: 1.59E5Base Peak F: ms2 MS b667JWUrine_HCDIT_CIDOTtop2_500ms

NL: 2.93E4Base Peak F: ms3 MS b667JWUrine_HCDIT_CIDOTtop2_500ms

3.1791 3.1891

MS 0.6 sec

CID MS3 OT

60 80 100 120 140 160 180m/z

0

20

40

60

80

100

124.0153

105.0077

180.9644141.9145

95.077369.0120

Theophylline MS3

isomers

Library

Match score 936.2

∆ 1.8 ppm ∆ 1.9 ppm Query

HCD MS2 IT

HCD MS2 IT

CID MS3 OT

O

N

NH NH

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The Industry’s Leading Portfolio of MS Solutions

HR/AM

MS, MSn

Appl

ied

Mar

kets

Research

Markets

Ion Traps Triple Quads

Tribrid Orbitrap MS

Non-targeted Analysis

Targeted Analysis

Quantitative Qualitative

• Biomarker Discovery • Proteomics • Metabolism

• Metabolomics • Proteomics • Bioanalysis

• Food Safety • Environmental • Clinical/Toxicology

• Metabolomics • PTM Analysis • Lipidomics

Transform Your Science

Exactive Series MS

29

Transform Your Science

new mass spectrometry tools to transform metabolomics and ... · 2 omics & the central dogma of...

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