1 The world leader in serving science
July.3.2013 Ken Miller Vice President of Marketing,
Life Sciences Mass Spectrometry
New Mass Spectrometry Tools to Transform Metabolomics and Lipidomics
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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!
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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
Orbitrap Fusion Tribrid Mass Spectrometer
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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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Orbitrap Fusion Tribrid Mass Spectrometer
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+
Internal Calibration: LC/MS of Omeprazole Metabolites
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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
Internal Calibration: LC/MS of Omeprazole Metabolites
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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
Internal Calibration: LC/MS of Omeprazole Metabolites
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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)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
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
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
146.06033
118.06544
132.08105 188.07079170.06030
MS HCD MS/MS
80 100 120 140 160 180 2000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
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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Orbitrap Fusion Tribrid Mass Spectrometer
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
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R=120k
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R=60k
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R=240k
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
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
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
150.0584R=627006
151.0618R=621502
152.0542R=601802
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
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
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
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
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
151.0617 C4
13CH12O2NS
151.0625 C5H12O17ONS 151.0646 C5
13C14CH918O17O
151.0577 C5H12O2N33S
151.554 14C4H10
18O233S
ASMS 2013 poster MP045 by Junhua Wang et al.
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160 165 170 175 180 185 190 195 200 205 210 215 2200
20
40
60
80
100
Rel
ativ
e A
bund
ance
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
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0573R=67606
179.050 179.055 179.060 179.0650
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=148204
179.0558R=130000
179.050 179.055 179.060 179.0650
10
20
30
40
50
60
70
80
90
100R
elat
ive
Abu
ndan
ce
179.0574R=304604
179.0560R=275000
179.050 179.055 179.060 179.0650
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
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
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.05763R=147660
179.055 179.060m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.05759R=280705
179.055 179.060m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.05747R=72040
179.055 179.060m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
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?
ASMS 2013 poster MP045 by Junhua Wang et al.
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Orbitrap Fusion Tribrid Mass Spectrometer
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)
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
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
ativ
e A
bund
ance
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
10
20
0
10
20
Rel
ativ
e A
bund
ance 0
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
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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
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
96.0556
69.0447
60 80 100 120 140 160 180 m/z
0
20
40
60
80
100
Rel
ativ
e Ab
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)
0
10
20
0
10
20
Rel
ativ
e A
bund
ance 0
10
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
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Transform Your Science
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