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A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library
Junhua Wang,1 David A. Peake,1 Robert Mistrik,2 Yingying Huang1
1Thermo Fisher Scientific Inc, San Jose, CA, USA; 2HighChem, Ltd., Bratislava, Slovakia
2 A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library
60 80 100 120m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
96.0556
69.0447
RT: 3.1775 - 3.1907
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 3.1891Full Scan MS
MS2
MS3
0.6 sec
HCDIT-MS2
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
CIDOT-MS3
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
An Improved Metabolomics WorkflowA traditional workflow for metabolite profiling and identification is based on a full scan LC/MS experiment followed by targeted MS/MS confirmation.
The major problems associated with this methodology are:1) Instrumentation limitations
• Does not allow simultaneous profiling and MSn in a single run, the scan speed su ers.
• Identification is usually based on accurate mass and MS2, but a large number of LC peaks remain not identifiable .
2) MS/MS library limitations• MS/MS spectral search is usually manually performed and time-
consuming; • Automated scoring for spectral matching is usually not available;• The fragmentation needs to be manually interpreted, which is not
only time-consuming but also prone to errors.
3) There is no good solution for unknowns.
The Orbitrap Fusion MS enables fast parallel data acquisition on a UHPLC timescale with a variety of dissociation choices The Orbitrap Fusion MS o ers ultimate flexibility in fragmentation methods at all stages of MSn analysis, with fragment ions detected by either mass analyzer. CID-HCD, HCD-CID, and HCD-HCD can all be set up as simple as the conventional CID-CID. This complete flexibility facilitates the examination of fragmentation pathways that leads to the most comprehensive structural information.
mzCloud MSn library enables MSn search for both high and low resolution mzCloud is a multistage MS library. Based on the spectral tree similarity, an algorithm called precursor ion fingerprinting (PIF) enables the identification of compounds even if they are not in the library, and the fragmental peaks can be annotated [2]. Powered by the novel MS and the mzCloud library, an improved, more connected workflow is shown below:
Major benefits of the improved workflow: 1) HCD MS/MS can be performed simultaneously with full scan MS on the
UHPLC timescale with ion trap detection at >22 Hz.2) Simultaneous ddMSn ion tree with flexible combinations of CID and/or HCD
fragmentation with ion trap and/or OrbitrapTM detection. 2) Both the MS2 and MSn will be automatically searched in mzCloud, the
matching entry will be scored based on PIF, and the fragments will be annotated.
4) De novo structural identification is enabled by using this workflow.
OverviewPurpose: To demonstrate the capability of the Thermo Scientific™ Orbitrap Fusion™ Tribrid™ Mass Spectrometer in combination with MSn library for metabolite structural identification.
Methods: UHPLC coupled to the Orbitrap Fusion MS to simultaneously perform global profiling and structural elucidation with MSn.
Results: Within UHPLC timescale (2-3 second FWHM), >10 cycles can be finished with fast ion trap HCD MS2 for top 11 precursors, each with ion trees of MS2-MS3(top2)-MS4(top2).
IntroductionThe structural identification of metabolites represents a significant challenge in metabolomics study. Multistage mass spectrometry (MSn) is a powerful tool for compound identification and structural elucidation that goes beyond identifying the exact same compounds, to discovering additional compounds with structural homology to those in the library. Combining with high resolution accurate mass (HRAM) measurement, MSn is highly e ective in identifying the unknown but biologically relevant compounds in metabolomics studies[1]. However, the speed on current platforms is yet to be e ectively compatible with UHPLC front end, which limited such application in biological samples.
Presented here is a new platform to identify endogenous metabolites using a novel high performance Orbitrap hybrid instrument in conjunction with UHPLC and an MSn library.
Methods Sample Preparation
Urine samples from an adult male were analyzed. The urine samples were diluted 1 to 10 with water and processed with a 3kD MWCO filter.
For plasma samples, proteins were removed with cold methanol and centrifugation, followed by centrifugal evaporation at 35 oC. The residue was reconstituted in MeOH/Water, 1/9, and also processed with a 3kD MWCO filter.
Liquid ChromatographyUHPLC separation was implemented on a Dionex Ultimate 3000 HPG (high-pressure gradient) pump using Hypersil GOLD RP C18 column at 450 L/min, column temperature at 55 oC. LC solvents were 0.1% FA (A) and 0.1% FA in MeOH (B). A linear gradient was applied from 0.5-50% B for 5.5 min, followed by increasing to 98% at 6 min, held 98% B for 6 min, then decreased to 0.5% at 13min, and equilibrated for another 2 min.
FIGURE 1. Schematic of Orbitrap Fusion Tribrid Mass Spectrometer
A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library Junhua Wang1, David A. Peake1, Robert Mistrik2, Yingying Huang11Thermo Fisher Scientific Inc, San Jose, CA; 2HighChem, Ltd., Bratislava, Slovakia
Results
S-Lens
Active beam guide (ABG)
Ultra-high-field Orbitrap mass analyzer
Large-surface-Area detector (LSAD)
Ion-routing multipole
Dual-pressure linear ion trap
Quadrupole mass filter
C-Trap
EASY-Internal Calibration ion source
FIGURE 2. Ultrahigh resolution resolves the isotopes
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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100
Rel
ativ
e A
bund
ance
150.0584R=627006
151.0618R=621502
152.0542R=601802
PositiveMode20130518092831 #654 RT: 2.60 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #539 RT: 1.44 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #511 RT: 1.33 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #615 RT: 1.97 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #237 RT: 0.51 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R=30k
R=60k
R=120k! =240kR=500k
L-Methionine C5H11NO2S
0.7ppm
Fine isotope pattern
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
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60
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Rel
ativ
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bund
ance
150.0583R=499553
151.0617R=499368
152.0541R=499443
PositiveSimulated
R=500,000
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
151.0617C4
13CH12O2NS
151.0625C5H12O17ONS
151.0646C5
13C14CH918O17O
151.0577C5H12O2N33S
151.55414C4H10
18O233S
PositiveObserved
Negative20130518111518 #1449 RT: 8.07 AV: 1 NL: 1.00E6T: FTMS - p ESI Full ms [140.00-220.00]
179 180 181 182m/z
0
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Rel
ativ
e A
bund
ance
179.0576R=550402
180.0611R=330000
NegativeFull Scan
0.55 ppm
Negative20130518111518 #158 RT: 1.66 AV: 1 NL: 1.24E6T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
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100
Rel
ativ
e A
bund
ance
179.0573R=67606
Negative20130518111518 #110 RT: 1.34 AV: 1 NL: 8.48E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
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Rel
ativ
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bund
ance
179.0574R=148204
179.0558R=130000
Negative20130518111518 #54 RT: 0.83 AV: 1 NL: 9.54E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
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30
40
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Rel
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bund
ance
179.0574R=304604
179.0560R=275000
Negative20130518111518 #21 RT: 0.40 AV: 1 NL: 7.38E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
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30
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Rel
ativ
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bund
ance
179.0574R=607806
179.0560R=562802
R=60,000 R=120,000
R=240,000 R=500,000200 M
1 M
1.1 ppm
0.56 ppm
0.55 ppm
0.55 ppm
0.56 ppm
1.7 ppm
0.55 ppm
FIGURE 3. Ultrahigh resolution resolves isobaric metabolites
RT: 2.75160 - 2.86730
2.76 2.77 2.78 2.79 2.80 2.81 2.82 2.83 2.84 2.85 2.86Time (min)
0
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60
70
80
90
1000
10
20
30
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50
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Rel
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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
NL:3.23E7Base Peak m/z= 205.09525-205.09935 F: ms MS JWUrine_FS240k_LM_Re
NL:2.85E7Base Peak m/z= 205.09525-205.09935 MS jwurine_fs240k_nolm_re
PBUrine_MS2_120k_600ms_bothLM #824 RT: 2.82 AV: 1 NL: 8.68E6F: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
80 100 120 140 160 180 200m/z
0
10
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40
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Rel
ativ
e A
bund
ance
146.06033
118.06544
132.08105 188.07079170.06030without Int. Cal.Avg =1.5 ppm
Full Scan MS HCD MS/MSJWUrine_MS2_120k_600ms_bothLM #896 RT: 2.84 AV: 1 NL: 1.08E7T: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
80 100 120 140 160 180 200m/z
0
10
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30
40
50
60
70
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100
Rel
ativ
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bund
ance
146.06020
118.06530
132.08090 170.06015 188.0706391.05422
0.2 ppmO
NH
NH
OHO
NH
0.9 ppm 0.7 ppmNH •
0.4 ppm
0.8 ppm
N2H
1 ppm
NH
N 2HNH
-0.1 ppm
with Int. Cal.Avg = 0.4 ppm
1.1 ppm
1.8 ppm
2.0 ppm
1.7 ppm
1.9ppm
2.9 ppm
1.1 ppm
with Int. Cal.
without Int. Cal.
Metabolite Identification Requires High Resolution and Isotope Ratio Measurements Structure elucidation of unknown small molecules by mass spectrometry is a great challenge. The first crucial step is to obtain correct elemental compositions.
Natural occurring elements can be monoisotopic (F, Na, P, I) or polyisotopic (H, C, N, O, S, Cl, Br) [1]. The molecule L-Methionine C5H11NO2S contains multiple polyisotopic elements, especially the S, which result in multiple isotopic peaks needing resolving power above 240,000 at m/z 200 (FWHM). Figure 2A shows the better isotope pattern of molecular ion [M+1+H]+. With increasing resolution up to 450,000 (FWHM), all five isotope peaks are clearly resolved. Figure 2B shows the simulated isotope pattern.
A
B
Metabolite Identification and Quantitation in Complex Samples Requires High ResolutionD-Glucose C6H12O6 and Paraxanthine C7H8N4O2 co-exist in human blood at distinct concentration levels: 5300 M (D-Glucose) vs. 10 M (Paraxanthine) [3]. Although D-Glucose is hundreds of fold more concentrated, it is less readily detectable because of its low ionization e ciency. Their masses di erentiate by 0.0013 amu (179.0561 vs. 179.0574), making the detection of these individual components very challenging. Figure 3 shows that, with >300,000 resolving power, the 200 M (D-Glucose) and 1 M (Paraxanthine) can be readily separated and quantified.
FIGURE 4. Easy Internal Calibration
Metabolite Identification Requires High Accuracy of Mass Measurement in Both MS Full Scan Level and MS/MS LevelFor an unknown metabolite, the number of calculated chemically possible formulae could be hundreds (20 ppm tolerance window), but the possibilities significantly reduce to tens (3 ppm) and several if the tolerance window is narrowed to sub-ppm. However, due to the large amount of known isomeric structures, one single formula may correspond to hundreds of structural isomers, thus structure identification of metabolite isomer using only MS full scan is not feasible. MS/MS and MSn techniques can help solve this analytical problem.
The Orbitrap Fusion MS has a built-in internal calibration functionality (Easy IC). Figure 4 shows LC-MS/MS data of L-Tryptophan from an urine sample with and without internal calibration. Sub-ppm mass accuracy was observed for MS and MS2 fragments with IC activated.
FIGURE 5. Rapid Ion Tree Acquisition on UHPLC Timescale from a Urine Sample (1 MS, 2 MS2 and 4 MS3 in 0.6sec/cycle).(A) Ion trap HCD MS2 followed by Orbitrap CID MS3 were acquired for the precursor ion m/z 181.0721. (B) Spectral tree search in mzCloud matches the MS3 with MS3 from theophylline with the highest score, indicating the unknown must have the same substructure as theophylline.
ConclusionThe Orbitrap Fusion MS o ers ultra high resolution and high fidelity in isotope ratio measurements of metabolites. This allows metabolites in complex mixture with a wide dynamic range be resolved and detected. The Easy-Internal Calibration allows compound assignment be readily done with confidence. The Orbitrap Fusion MS provides ultimate flexibility that facilitates the examination of multiple structural pathways for more structural information at higher speed.
The mzCloud library allows metabolite identification with MS2 and/or MSn data in an automated fashion. The identification based on multistage MS spectral trees and PIF algorithm opens the possibility to identify unknown metabolites and isomers. An improved metabolomics workflow is enabled.
References1. Budzikiewicz H, Grigsby RD, Mass Spectrom. Rev. 2006, 25(1):146-157.
2. Sheldon MT, Mistrik R, Croley TR, J. Am. Soc. Mass Spectrom. 2009, 20(3):370-376
3. http://www.hmdb.ca/metabolites/HMDB00122 & HMDB01860
A
BTheophylline
MS3
isomers
Query (down) and library spectra (up) matchScore
N
NH NH
N
NH
1.5 ppm 1.5 ppm
OH
NH
1 ppm
2.1 ppm
FIGURE 1. The Orbitrap Fusion MS and the Drag and Drop Instrument Method Editor.
O
N
NH NH
3Thermo Scientific Poster Note • PN ASMS13_MP 045_JWang e 06/13S
60 80 100 120m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
96.0556
69.0447
RT: 3.1775 - 3.1907
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 3.1891Full Scan MS
MS2
MS3
0.6 sec
HCDIT-MS2
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
CIDOT-MS3
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
An Improved Metabolomics WorkflowA traditional workflow for metabolite profiling and identification is based on a full scan LC/MS experiment followed by targeted MS/MS confirmation.
The major problems associated with this methodology are:1) Instrumentation limitations
• Does not allow simultaneous profiling and MSn in a single run, the scan speed su ers.
• Identification is usually based on accurate mass and MS2, but a large number of LC peaks remain not identifiable .
2) MS/MS library limitations• MS/MS spectral search is usually manually performed and time-
consuming; • Automated scoring for spectral matching is usually not available;• The fragmentation needs to be manually interpreted, which is not
only time-consuming but also prone to errors.
3) There is no good solution for unknowns.
The Orbitrap Fusion MS enables fast parallel data acquisition on a UHPLC timescale with a variety of dissociation choices The Orbitrap Fusion MS o ers ultimate flexibility in fragmentation methods at all stages of MSn analysis, with fragment ions detected by either mass analyzer. CID-HCD, HCD-CID, and HCD-HCD can all be set up as simple as the conventional CID-CID. This complete flexibility facilitates the examination of fragmentation pathways that leads to the most comprehensive structural information.
mzCloud MSn library enables MSn search for both high and low resolution mzCloud is a multistage MS library. Based on the spectral tree similarity, an algorithm called precursor ion fingerprinting (PIF) enables the identification of compounds even if they are not in the library, and the fragmental peaks can be annotated [2]. Powered by the novel MS and the mzCloud library, an improved, more connected workflow is shown below:
Major benefits of the improved workflow: 1) HCD MS/MS can be performed simultaneously with full scan MS on the
UHPLC timescale with ion trap detection at >22 Hz.2) Simultaneous ddMSn ion tree with flexible combinations of CID and/or HCD
fragmentation with ion trap and/or OrbitrapTM detection. 2) Both the MS2 and MSn will be automatically searched in mzCloud, the
matching entry will be scored based on PIF, and the fragments will be annotated.
4) De novo structural identification is enabled by using this workflow.
OverviewPurpose: To demonstrate the capability of the Thermo Scientific™ Orbitrap Fusion™ Tribrid™ Mass Spectrometer in combination with MSn library for metabolite structural identification.
Methods: UHPLC coupled to the Orbitrap Fusion MS to simultaneously perform global profiling and structural elucidation with MSn.
Results: Within UHPLC timescale (2-3 second FWHM), >10 cycles can be finished with fast ion trap HCD MS2 for top 11 precursors, each with ion trees of MS2-MS3(top2)-MS4(top2).
IntroductionThe structural identification of metabolites represents a significant challenge in metabolomics study. Multistage mass spectrometry (MSn) is a powerful tool for compound identification and structural elucidation that goes beyond identifying the exact same compounds, to discovering additional compounds with structural homology to those in the library. Combining with high resolution accurate mass (HRAM) measurement, MSn is highly e ective in identifying the unknown but biologically relevant compounds in metabolomics studies[1]. However, the speed on current platforms is yet to be e ectively compatible with UHPLC front end, which limited such application in biological samples.
Presented here is a new platform to identify endogenous metabolites using a novel high performance Orbitrap hybrid instrument in conjunction with UHPLC and an MSn library.
Methods Sample Preparation
Urine samples from an adult male were analyzed. The urine samples were diluted 1 to 10 with water and processed with a 3kD MWCO filter.
For plasma samples, proteins were removed with cold methanol and centrifugation, followed by centrifugal evaporation at 35 oC. The residue was reconstituted in MeOH/Water, 1/9, and also processed with a 3kD MWCO filter.
Liquid ChromatographyUHPLC separation was implemented on a Dionex Ultimate 3000 HPG (high-pressure gradient) pump using Hypersil GOLD RP C18 column at 450 L/min, column temperature at 55 oC. LC solvents were 0.1% FA (A) and 0.1% FA in MeOH (B). A linear gradient was applied from 0.5-50% B for 5.5 min, followed by increasing to 98% at 6 min, held 98% B for 6 min, then decreased to 0.5% at 13min, and equilibrated for another 2 min.
FIGURE 1. Schematic of Orbitrap Fusion Tribrid Mass Spectrometer
A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library Junhua Wang1, David A. Peake1, Robert Mistrik2, Yingying Huang11Thermo Fisher Scientific Inc, San Jose, CA; 2HighChem, Ltd., Bratislava, Slovakia
Results
S-Lens
Active beam guide (ABG)
Ultra-high-field Orbitrap mass analyzer
Large-surface-Area detector (LSAD)
Ion-routing multipole
Dual-pressure linear ion trap
Quadrupole mass filter
C-Trap
EASY-Internal Calibration ion source
FIGURE 2. Ultrahigh resolution resolves the isotopes
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
PositiveMode20130518092831 #654 RT: 2.60 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #539 RT: 1.44 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #511 RT: 1.33 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #615 RT: 1.97 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #237 RT: 0.51 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R=30k
R=60k
R=120k! =240kR=500k
L-Methionine C5H11NO2S
0.7ppm
Fine isotope pattern
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
PositiveSimulated
R=500,000
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
151.0617C4
13CH12O2NS
151.0625C5H12O17ONS
151.0646C5
13C14CH918O17O
151.0577C5H12O2N33S
151.55414C4H10
18O233S
PositiveObserved
Negative20130518111518 #1449 RT: 8.07 AV: 1 NL: 1.00E6T: FTMS - p ESI Full ms [140.00-220.00]
179 180 181 182m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0576R=550402
180.0611R=330000
NegativeFull Scan
0.55 ppm
Negative20130518111518 #158 RT: 1.66 AV: 1 NL: 1.24E6T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0573R=67606
Negative20130518111518 #110 RT: 1.34 AV: 1 NL: 8.48E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=148204
179.0558R=130000
Negative20130518111518 #54 RT: 0.83 AV: 1 NL: 9.54E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=304604
179.0560R=275000
Negative20130518111518 #21 RT: 0.40 AV: 1 NL: 7.38E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
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=500,000200 M
1 M
1.1 ppm
0.56 ppm
0.55 ppm
0.55 ppm
0.56 ppm
1.7 ppm
0.55 ppm
FIGURE 3. Ultrahigh resolution resolves isobaric metabolites
RT: 2.75160 - 2.86730
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
NL:3.23E7Base Peak m/z= 205.09525-205.09935 F: ms MS JWUrine_FS240k_LM_Re
NL:2.85E7Base Peak m/z= 205.09525-205.09935 MS jwurine_fs240k_nolm_re
PBUrine_MS2_120k_600ms_bothLM #824 RT: 2.82 AV: 1 NL: 8.68E6F: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
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 188.07079170.06030without Int. Cal.Avg =1.5 ppm
Full Scan MS HCD MS/MSJWUrine_MS2_120k_600ms_bothLM #896 RT: 2.84 AV: 1 NL: 1.08E7T: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
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
146.06020
118.06530
132.08090 170.06015 188.0706391.05422
0.2 ppmO
NH
NH
OHO
NH
0.9 ppm 0.7 ppmNH •
0.4 ppm
0.8 ppm
N2H
1 ppm
NH
N 2HNH
-0.1 ppm
with Int. Cal.Avg = 0.4 ppm
1.1 ppm
1.8 ppm
2.0 ppm
1.7 ppm
1.9ppm
2.9 ppm
1.1 ppm
with Int. Cal.
without Int. Cal.
Metabolite Identification Requires High Resolution and Isotope Ratio Measurements Structure elucidation of unknown small molecules by mass spectrometry is a great challenge. The first crucial step is to obtain correct elemental compositions.
Natural occurring elements can be monoisotopic (F, Na, P, I) or polyisotopic (H, C, N, O, S, Cl, Br) [1]. The molecule L-Methionine C5H11NO2S contains multiple polyisotopic elements, especially the S, which result in multiple isotopic peaks needing resolving power above 240,000 at m/z 200 (FWHM). Figure 2A shows the better isotope pattern of molecular ion [M+1+H]+. With increasing resolution up to 450,000 (FWHM), all five isotope peaks are clearly resolved. Figure 2B shows the simulated isotope pattern.
A
B
Metabolite Identification and Quantitation in Complex Samples Requires High ResolutionD-Glucose C6H12O6 and Paraxanthine C7H8N4O2 co-exist in human blood at distinct concentration levels: 5300 M (D-Glucose) vs. 10 M (Paraxanthine) [3]. Although D-Glucose is hundreds of fold more concentrated, it is less readily detectable because of its low ionization e ciency. Their masses di erentiate by 0.0013 amu (179.0561 vs. 179.0574), making the detection of these individual components very challenging. Figure 3 shows that, with >300,000 resolving power, the 200 M (D-Glucose) and 1 M (Paraxanthine) can be readily separated and quantified.
FIGURE 4. Easy Internal Calibration
Metabolite Identification Requires High Accuracy of Mass Measurement in Both MS Full Scan Level and MS/MS LevelFor an unknown metabolite, the number of calculated chemically possible formulae could be hundreds (20 ppm tolerance window), but the possibilities significantly reduce to tens (3 ppm) and several if the tolerance window is narrowed to sub-ppm. However, due to the large amount of known isomeric structures, one single formula may correspond to hundreds of structural isomers, thus structure identification of metabolite isomer using only MS full scan is not feasible. MS/MS and MSn techniques can help solve this analytical problem.
The Orbitrap Fusion MS has a built-in internal calibration functionality (Easy IC). Figure 4 shows LC-MS/MS data of L-Tryptophan from an urine sample with and without internal calibration. Sub-ppm mass accuracy was observed for MS and MS2 fragments with IC activated.
FIGURE 5. Rapid Ion Tree Acquisition on UHPLC Timescale from a Urine Sample (1 MS, 2 MS2 and 4 MS3 in 0.6sec/cycle).(A) Ion trap HCD MS2 followed by Orbitrap CID MS3 were acquired for the precursor ion m/z 181.0721. (B) Spectral tree search in mzCloud matches the MS3 with MS3 from theophylline with the highest score, indicating the unknown must have the same substructure as theophylline.
ConclusionThe Orbitrap Fusion MS o ers ultra high resolution and high fidelity in isotope ratio measurements of metabolites. This allows metabolites in complex mixture with a wide dynamic range be resolved and detected. The Easy-Internal Calibration allows compound assignment be readily done with confidence. The Orbitrap Fusion MS provides ultimate flexibility that facilitates the examination of multiple structural pathways for more structural information at higher speed.
The mzCloud library allows metabolite identification with MS2 and/or MSn data in an automated fashion. The identification based on multistage MS spectral trees and PIF algorithm opens the possibility to identify unknown metabolites and isomers. An improved metabolomics workflow is enabled.
References1. Budzikiewicz H, Grigsby RD, Mass Spectrom. Rev. 2006, 25(1):146-157.
2. Sheldon MT, Mistrik R, Croley TR, J. Am. Soc. Mass Spectrom. 2009, 20(3):370-376
3. http://www.hmdb.ca/metabolites/HMDB00122 & HMDB01860
A
BTheophylline
MS3
isomers
Query (down) and library spectra (up) matchScore
N
NH NH
N
NH
1.5 ppm 1.5 ppm
OH
NH
1 ppm
2.1 ppm
FIGURE 1. The Orbitrap Fusion MS and the Drag and Drop Instrument Method Editor.
O
N
NH NH
4 A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library
60 80 100 120m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
96.0556
69.0447
RT: 3.1775 - 3.1907
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 3.1891Full Scan MS
MS2
MS3
0.6 sec
HCDIT-MS2
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
CIDOT-MS3
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
An Improved Metabolomics WorkflowA traditional workflow for metabolite profiling and identification is based on a full scan LC/MS experiment followed by targeted MS/MS confirmation.
The major problems associated with this methodology are:1) Instrumentation limitations
• Does not allow simultaneous profiling and MSn in a single run, the scan speed su ers.
• Identification is usually based on accurate mass and MS2, but a large number of LC peaks remain not identifiable .
2) MS/MS library limitations• MS/MS spectral search is usually manually performed and time-
consuming; • Automated scoring for spectral matching is usually not available;• The fragmentation needs to be manually interpreted, which is not
only time-consuming but also prone to errors.
3) There is no good solution for unknowns.
The Orbitrap Fusion MS enables fast parallel data acquisition on a UHPLC timescale with a variety of dissociation choices The Orbitrap Fusion MS o ers ultimate flexibility in fragmentation methods at all stages of MSn analysis, with fragment ions detected by either mass analyzer. CID-HCD, HCD-CID, and HCD-HCD can all be set up as simple as the conventional CID-CID. This complete flexibility facilitates the examination of fragmentation pathways that leads to the most comprehensive structural information.
mzCloud MSn library enables MSn search for both high and low resolution mzCloud is a multistage MS library. Based on the spectral tree similarity, an algorithm called precursor ion fingerprinting (PIF) enables the identification of compounds even if they are not in the library, and the fragmental peaks can be annotated [2]. Powered by the novel MS and the mzCloud library, an improved, more connected workflow is shown below:
Major benefits of the improved workflow: 1) HCD MS/MS can be performed simultaneously with full scan MS on the
UHPLC timescale with ion trap detection at >22 Hz.2) Simultaneous ddMSn ion tree with flexible combinations of CID and/or HCD
fragmentation with ion trap and/or OrbitrapTM detection. 2) Both the MS2 and MSn will be automatically searched in mzCloud, the
matching entry will be scored based on PIF, and the fragments will be annotated.
4) De novo structural identification is enabled by using this workflow.
OverviewPurpose: To demonstrate the capability of the Thermo Scientific™ Orbitrap Fusion™ Tribrid™ Mass Spectrometer in combination with MSn library for metabolite structural identification.
Methods: UHPLC coupled to the Orbitrap Fusion MS to simultaneously perform global profiling and structural elucidation with MSn.
Results: Within UHPLC timescale (2-3 second FWHM), >10 cycles can be finished with fast ion trap HCD MS2 for top 11 precursors, each with ion trees of MS2-MS3(top2)-MS4(top2).
IntroductionThe structural identification of metabolites represents a significant challenge in metabolomics study. Multistage mass spectrometry (MSn) is a powerful tool for compound identification and structural elucidation that goes beyond identifying the exact same compounds, to discovering additional compounds with structural homology to those in the library. Combining with high resolution accurate mass (HRAM) measurement, MSn is highly e ective in identifying the unknown but biologically relevant compounds in metabolomics studies[1]. However, the speed on current platforms is yet to be e ectively compatible with UHPLC front end, which limited such application in biological samples.
Presented here is a new platform to identify endogenous metabolites using a novel high performance Orbitrap hybrid instrument in conjunction with UHPLC and an MSn library.
Methods Sample Preparation
Urine samples from an adult male were analyzed. The urine samples were diluted 1 to 10 with water and processed with a 3kD MWCO filter.
For plasma samples, proteins were removed with cold methanol and centrifugation, followed by centrifugal evaporation at 35 oC. The residue was reconstituted in MeOH/Water, 1/9, and also processed with a 3kD MWCO filter.
Liquid ChromatographyUHPLC separation was implemented on a Dionex Ultimate 3000 HPG (high-pressure gradient) pump using Hypersil GOLD RP C18 column at 450 L/min, column temperature at 55 oC. LC solvents were 0.1% FA (A) and 0.1% FA in MeOH (B). A linear gradient was applied from 0.5-50% B for 5.5 min, followed by increasing to 98% at 6 min, held 98% B for 6 min, then decreased to 0.5% at 13min, and equilibrated for another 2 min.
FIGURE 1. Schematic of Orbitrap Fusion Tribrid Mass Spectrometer
A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library Junhua Wang1, David A. Peake1, Robert Mistrik2, Yingying Huang11Thermo Fisher Scientific Inc, San Jose, CA; 2HighChem, Ltd., Bratislava, Slovakia
Results
S-Lens
Active beam guide (ABG)
Ultra-high-field Orbitrap mass analyzer
Large-surface-Area detector (LSAD)
Ion-routing multipole
Dual-pressure linear ion trap
Quadrupole mass filter
C-Trap
EASY-Internal Calibration ion source
FIGURE 2. Ultrahigh resolution resolves the isotopes
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
PositiveMode20130518092831 #654 RT: 2.60 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #539 RT: 1.44 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #511 RT: 1.33 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #615 RT: 1.97 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #237 RT: 0.51 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R=30k
R=60k
R=120k! =240kR=500k
L-Methionine C5H11NO2S
0.7ppm
Fine isotope pattern
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
PositiveSimulated
R=500,000
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
151.0617C4
13CH12O2NS
151.0625C5H12O17ONS
151.0646C5
13C14CH918O17O
151.0577C5H12O2N33S
151.55414C4H10
18O233S
PositiveObserved
Negative20130518111518 #1449 RT: 8.07 AV: 1 NL: 1.00E6T: FTMS - p ESI Full ms [140.00-220.00]
179 180 181 182m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0576R=550402
180.0611R=330000
NegativeFull Scan
0.55 ppm
Negative20130518111518 #158 RT: 1.66 AV: 1 NL: 1.24E6T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0573R=67606
Negative20130518111518 #110 RT: 1.34 AV: 1 NL: 8.48E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=148204
179.0558R=130000
Negative20130518111518 #54 RT: 0.83 AV: 1 NL: 9.54E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=304604
179.0560R=275000
Negative20130518111518 #21 RT: 0.40 AV: 1 NL: 7.38E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
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=500,000200 M
1 M
1.1 ppm
0.56 ppm
0.55 ppm
0.55 ppm
0.56 ppm
1.7 ppm
0.55 ppm
FIGURE 3. Ultrahigh resolution resolves isobaric metabolites
RT: 2.75160 - 2.86730
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
NL:3.23E7Base Peak m/z= 205.09525-205.09935 F: ms MS JWUrine_FS240k_LM_Re
NL:2.85E7Base Peak m/z= 205.09525-205.09935 MS jwurine_fs240k_nolm_re
PBUrine_MS2_120k_600ms_bothLM #824 RT: 2.82 AV: 1 NL: 8.68E6F: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
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 188.07079170.06030without Int. Cal.Avg =1.5 ppm
Full Scan MS HCD MS/MSJWUrine_MS2_120k_600ms_bothLM #896 RT: 2.84 AV: 1 NL: 1.08E7T: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
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
146.06020
118.06530
132.08090 170.06015 188.0706391.05422
0.2 ppmO
NH
NH
OHO
NH
0.9 ppm 0.7 ppmNH •
0.4 ppm
0.8 ppm
N2H
1 ppm
NH
N 2HNH
-0.1 ppm
with Int. Cal.Avg = 0.4 ppm
1.1 ppm
1.8 ppm
2.0 ppm
1.7 ppm
1.9ppm
2.9 ppm
1.1 ppm
with Int. Cal.
without Int. Cal.
Metabolite Identification Requires High Resolution and Isotope Ratio Measurements Structure elucidation of unknown small molecules by mass spectrometry is a great challenge. The first crucial step is to obtain correct elemental compositions.
Natural occurring elements can be monoisotopic (F, Na, P, I) or polyisotopic (H, C, N, O, S, Cl, Br) [1]. The molecule L-Methionine C5H11NO2S contains multiple polyisotopic elements, especially the S, which result in multiple isotopic peaks needing resolving power above 240,000 at m/z 200 (FWHM). Figure 2A shows the better isotope pattern of molecular ion [M+1+H]+. With increasing resolution up to 450,000 (FWHM), all five isotope peaks are clearly resolved. Figure 2B shows the simulated isotope pattern.
A
B
Metabolite Identification and Quantitation in Complex Samples Requires High ResolutionD-Glucose C6H12O6 and Paraxanthine C7H8N4O2 co-exist in human blood at distinct concentration levels: 5300 M (D-Glucose) vs. 10 M (Paraxanthine) [3]. Although D-Glucose is hundreds of fold more concentrated, it is less readily detectable because of its low ionization e ciency. Their masses di erentiate by 0.0013 amu (179.0561 vs. 179.0574), making the detection of these individual components very challenging. Figure 3 shows that, with >300,000 resolving power, the 200 M (D-Glucose) and 1 M (Paraxanthine) can be readily separated and quantified.
FIGURE 4. Easy Internal Calibration
Metabolite Identification Requires High Accuracy of Mass Measurement in Both MS Full Scan Level and MS/MS LevelFor an unknown metabolite, the number of calculated chemically possible formulae could be hundreds (20 ppm tolerance window), but the possibilities significantly reduce to tens (3 ppm) and several if the tolerance window is narrowed to sub-ppm. However, due to the large amount of known isomeric structures, one single formula may correspond to hundreds of structural isomers, thus structure identification of metabolite isomer using only MS full scan is not feasible. MS/MS and MSn techniques can help solve this analytical problem.
The Orbitrap Fusion MS has a built-in internal calibration functionality (Easy IC). Figure 4 shows LC-MS/MS data of L-Tryptophan from an urine sample with and without internal calibration. Sub-ppm mass accuracy was observed for MS and MS2 fragments with IC activated.
FIGURE 5. Rapid Ion Tree Acquisition on UHPLC Timescale from a Urine Sample (1 MS, 2 MS2 and 4 MS3 in 0.6sec/cycle).(A) Ion trap HCD MS2 followed by Orbitrap CID MS3 were acquired for the precursor ion m/z 181.0721. (B) Spectral tree search in mzCloud matches the MS3 with MS3 from theophylline with the highest score, indicating the unknown must have the same substructure as theophylline.
ConclusionThe Orbitrap Fusion MS o ers ultra high resolution and high fidelity in isotope ratio measurements of metabolites. This allows metabolites in complex mixture with a wide dynamic range be resolved and detected. The Easy-Internal Calibration allows compound assignment be readily done with confidence. The Orbitrap Fusion MS provides ultimate flexibility that facilitates the examination of multiple structural pathways for more structural information at higher speed.
The mzCloud library allows metabolite identification with MS2 and/or MSn data in an automated fashion. The identification based on multistage MS spectral trees and PIF algorithm opens the possibility to identify unknown metabolites and isomers. An improved metabolomics workflow is enabled.
References1. Budzikiewicz H, Grigsby RD, Mass Spectrom. Rev. 2006, 25(1):146-157.
2. Sheldon MT, Mistrik R, Croley TR, J. Am. Soc. Mass Spectrom. 2009, 20(3):370-376
3. http://www.hmdb.ca/metabolites/HMDB00122 & HMDB01860
A
BTheophylline
MS3
isomers
Query (down) and library spectra (up) matchScore
N
NH NH
N
NH
1.5 ppm 1.5 ppm
OH
NH
1 ppm
2.1 ppm
FIGURE 1. The Orbitrap Fusion MS and the Drag and Drop Instrument Method Editor.
O
N
NH NH
5Thermo Scientific Poster Note • PN ASMS13_MP 045_JWang e 06/13S
60 80 100 120m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
96.0556
69.0447
RT: 3.1775 - 3.1907
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 3.1891Full Scan MS
MS2
MS3
0.6 sec
HCDIT-MS2
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
CIDOT-MS3
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
An Improved Metabolomics WorkflowA traditional workflow for metabolite profiling and identification is based on a full scan LC/MS experiment followed by targeted MS/MS confirmation.
The major problems associated with this methodology are:1) Instrumentation limitations
• Does not allow simultaneous profiling and MSn in a single run, the scan speed su ers.
• Identification is usually based on accurate mass and MS2, but a large number of LC peaks remain not identifiable .
2) MS/MS library limitations• MS/MS spectral search is usually manually performed and time-
consuming; • Automated scoring for spectral matching is usually not available;• The fragmentation needs to be manually interpreted, which is not
only time-consuming but also prone to errors.
3) There is no good solution for unknowns.
The Orbitrap Fusion MS enables fast parallel data acquisition on a UHPLC timescale with a variety of dissociation choices The Orbitrap Fusion MS o ers ultimate flexibility in fragmentation methods at all stages of MSn analysis, with fragment ions detected by either mass analyzer. CID-HCD, HCD-CID, and HCD-HCD can all be set up as simple as the conventional CID-CID. This complete flexibility facilitates the examination of fragmentation pathways that leads to the most comprehensive structural information.
mzCloud MSn library enables MSn search for both high and low resolution mzCloud is a multistage MS library. Based on the spectral tree similarity, an algorithm called precursor ion fingerprinting (PIF) enables the identification of compounds even if they are not in the library, and the fragmental peaks can be annotated [2]. Powered by the novel MS and the mzCloud library, an improved, more connected workflow is shown below:
Major benefits of the improved workflow: 1) HCD MS/MS can be performed simultaneously with full scan MS on the
UHPLC timescale with ion trap detection at >22 Hz.2) Simultaneous ddMSn ion tree with flexible combinations of CID and/or HCD
fragmentation with ion trap and/or OrbitrapTM detection. 2) Both the MS2 and MSn will be automatically searched in mzCloud, the
matching entry will be scored based on PIF, and the fragments will be annotated.
4) De novo structural identification is enabled by using this workflow.
OverviewPurpose: To demonstrate the capability of the Thermo Scientific™ Orbitrap Fusion™ Tribrid™ Mass Spectrometer in combination with MSn library for metabolite structural identification.
Methods: UHPLC coupled to the Orbitrap Fusion MS to simultaneously perform global profiling and structural elucidation with MSn.
Results: Within UHPLC timescale (2-3 second FWHM), >10 cycles can be finished with fast ion trap HCD MS2 for top 11 precursors, each with ion trees of MS2-MS3(top2)-MS4(top2).
IntroductionThe structural identification of metabolites represents a significant challenge in metabolomics study. Multistage mass spectrometry (MSn) is a powerful tool for compound identification and structural elucidation that goes beyond identifying the exact same compounds, to discovering additional compounds with structural homology to those in the library. Combining with high resolution accurate mass (HRAM) measurement, MSn is highly e ective in identifying the unknown but biologically relevant compounds in metabolomics studies[1]. However, the speed on current platforms is yet to be e ectively compatible with UHPLC front end, which limited such application in biological samples.
Presented here is a new platform to identify endogenous metabolites using a novel high performance Orbitrap hybrid instrument in conjunction with UHPLC and an MSn library.
Methods Sample Preparation
Urine samples from an adult male were analyzed. The urine samples were diluted 1 to 10 with water and processed with a 3kD MWCO filter.
For plasma samples, proteins were removed with cold methanol and centrifugation, followed by centrifugal evaporation at 35 oC. The residue was reconstituted in MeOH/Water, 1/9, and also processed with a 3kD MWCO filter.
Liquid ChromatographyUHPLC separation was implemented on a Dionex Ultimate 3000 HPG (high-pressure gradient) pump using Hypersil GOLD RP C18 column at 450 L/min, column temperature at 55 oC. LC solvents were 0.1% FA (A) and 0.1% FA in MeOH (B). A linear gradient was applied from 0.5-50% B for 5.5 min, followed by increasing to 98% at 6 min, held 98% B for 6 min, then decreased to 0.5% at 13min, and equilibrated for another 2 min.
FIGURE 1. Schematic of Orbitrap Fusion Tribrid Mass Spectrometer
A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library Junhua Wang1, David A. Peake1, Robert Mistrik2, Yingying Huang11Thermo Fisher Scientific Inc, San Jose, CA; 2HighChem, Ltd., Bratislava, Slovakia
Results
S-Lens
Active beam guide (ABG)
Ultra-high-field Orbitrap mass analyzer
Large-surface-Area detector (LSAD)
Ion-routing multipole
Dual-pressure linear ion trap
Quadrupole mass filter
C-Trap
EASY-Internal Calibration ion source
FIGURE 2. Ultrahigh resolution resolves the isotopes
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
PositiveMode20130518092831 #654 RT: 2.60 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #539 RT: 1.44 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #511 RT: 1.33 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #615 RT: 1.97 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #237 RT: 0.51 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R=30k
R=60k
R=120k! =240kR=500k
L-Methionine C5H11NO2S
0.7ppm
Fine isotope pattern
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
PositiveSimulated
R=500,000
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
151.0617C4
13CH12O2NS
151.0625C5H12O17ONS
151.0646C5
13C14CH918O17O
151.0577C5H12O2N33S
151.55414C4H10
18O233S
PositiveObserved
Negative20130518111518 #1449 RT: 8.07 AV: 1 NL: 1.00E6T: FTMS - p ESI Full ms [140.00-220.00]
179 180 181 182m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0576R=550402
180.0611R=330000
NegativeFull Scan
0.55 ppm
Negative20130518111518 #158 RT: 1.66 AV: 1 NL: 1.24E6T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0573R=67606
Negative20130518111518 #110 RT: 1.34 AV: 1 NL: 8.48E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=148204
179.0558R=130000
Negative20130518111518 #54 RT: 0.83 AV: 1 NL: 9.54E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=304604
179.0560R=275000
Negative20130518111518 #21 RT: 0.40 AV: 1 NL: 7.38E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
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=500,000200 M
1 M
1.1 ppm
0.56 ppm
0.55 ppm
0.55 ppm
0.56 ppm
1.7 ppm
0.55 ppm
FIGURE 3. Ultrahigh resolution resolves isobaric metabolites
RT: 2.75160 - 2.86730
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
NL:3.23E7Base Peak m/z= 205.09525-205.09935 F: ms MS JWUrine_FS240k_LM_Re
NL:2.85E7Base Peak m/z= 205.09525-205.09935 MS jwurine_fs240k_nolm_re
PBUrine_MS2_120k_600ms_bothLM #824 RT: 2.82 AV: 1 NL: 8.68E6F: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
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 188.07079170.06030without Int. Cal.Avg =1.5 ppm
Full Scan MS HCD MS/MSJWUrine_MS2_120k_600ms_bothLM #896 RT: 2.84 AV: 1 NL: 1.08E7T: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
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
146.06020
118.06530
132.08090 170.06015 188.0706391.05422
0.2 ppmO
NH
NH
OHO
NH
0.9 ppm 0.7 ppmNH •
0.4 ppm
0.8 ppm
N2H
1 ppm
NH
N 2HNH
-0.1 ppm
with Int. Cal.Avg = 0.4 ppm
1.1 ppm
1.8 ppm
2.0 ppm
1.7 ppm
1.9ppm
2.9 ppm
1.1 ppm
with Int. Cal.
without Int. Cal.
Metabolite Identification Requires High Resolution and Isotope Ratio Measurements Structure elucidation of unknown small molecules by mass spectrometry is a great challenge. The first crucial step is to obtain correct elemental compositions.
Natural occurring elements can be monoisotopic (F, Na, P, I) or polyisotopic (H, C, N, O, S, Cl, Br) [1]. The molecule L-Methionine C5H11NO2S contains multiple polyisotopic elements, especially the S, which result in multiple isotopic peaks needing resolving power above 240,000 at m/z 200 (FWHM). Figure 2A shows the better isotope pattern of molecular ion [M+1+H]+. With increasing resolution up to 450,000 (FWHM), all five isotope peaks are clearly resolved. Figure 2B shows the simulated isotope pattern.
A
B
Metabolite Identification and Quantitation in Complex Samples Requires High ResolutionD-Glucose C6H12O6 and Paraxanthine C7H8N4O2 co-exist in human blood at distinct concentration levels: 5300 M (D-Glucose) vs. 10 M (Paraxanthine) [3]. Although D-Glucose is hundreds of fold more concentrated, it is less readily detectable because of its low ionization e ciency. Their masses di erentiate by 0.0013 amu (179.0561 vs. 179.0574), making the detection of these individual components very challenging. Figure 3 shows that, with >300,000 resolving power, the 200 M (D-Glucose) and 1 M (Paraxanthine) can be readily separated and quantified.
FIGURE 4. Easy Internal Calibration
Metabolite Identification Requires High Accuracy of Mass Measurement in Both MS Full Scan Level and MS/MS LevelFor an unknown metabolite, the number of calculated chemically possible formulae could be hundreds (20 ppm tolerance window), but the possibilities significantly reduce to tens (3 ppm) and several if the tolerance window is narrowed to sub-ppm. However, due to the large amount of known isomeric structures, one single formula may correspond to hundreds of structural isomers, thus structure identification of metabolite isomer using only MS full scan is not feasible. MS/MS and MSn techniques can help solve this analytical problem.
The Orbitrap Fusion MS has a built-in internal calibration functionality (Easy IC). Figure 4 shows LC-MS/MS data of L-Tryptophan from an urine sample with and without internal calibration. Sub-ppm mass accuracy was observed for MS and MS2 fragments with IC activated.
FIGURE 5. Rapid Ion Tree Acquisition on UHPLC Timescale from a Urine Sample (1 MS, 2 MS2 and 4 MS3 in 0.6sec/cycle).(A) Ion trap HCD MS2 followed by Orbitrap CID MS3 were acquired for the precursor ion m/z 181.0721. (B) Spectral tree search in mzCloud matches the MS3 with MS3 from theophylline with the highest score, indicating the unknown must have the same substructure as theophylline.
ConclusionThe Orbitrap Fusion MS o ers ultra high resolution and high fidelity in isotope ratio measurements of metabolites. This allows metabolites in complex mixture with a wide dynamic range be resolved and detected. The Easy-Internal Calibration allows compound assignment be readily done with confidence. The Orbitrap Fusion MS provides ultimate flexibility that facilitates the examination of multiple structural pathways for more structural information at higher speed.
The mzCloud library allows metabolite identification with MS2 and/or MSn data in an automated fashion. The identification based on multistage MS spectral trees and PIF algorithm opens the possibility to identify unknown metabolites and isomers. An improved metabolomics workflow is enabled.
References1. Budzikiewicz H, Grigsby RD, Mass Spectrom. Rev. 2006, 25(1):146-157.
2. Sheldon MT, Mistrik R, Croley TR, J. Am. Soc. Mass Spectrom. 2009, 20(3):370-376
3. http://www.hmdb.ca/metabolites/HMDB00122 & HMDB01860
A
BTheophylline
MS3
isomers
Query (down) and library spectra (up) matchScore
N
NH NH
N
NH
1.5 ppm 1.5 ppm
OH
NH
1 ppm
2.1 ppm
FIGURE 1. The Orbitrap Fusion MS and the Drag and Drop Instrument Method Editor.
O
N
NH NH
6 A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library
60 80 100 120m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
96.0556
69.0447
RT: 3.1775 - 3.1907
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 3.1891Full Scan MS
MS2
MS3
0.6 sec
HCDIT-MS2
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
CIDOT-MS3
60 80 100 120 140 160 180m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
124.0153
105.0077
180.9644141.9145
95.077369.0120
An Improved Metabolomics WorkflowA traditional workflow for metabolite profiling and identification is based on a full scan LC/MS experiment followed by targeted MS/MS confirmation.
The major problems associated with this methodology are:1) Instrumentation limitations
• Does not allow simultaneous profiling and MSn in a single run, the scan speed su ers.
• Identification is usually based on accurate mass and MS2, but a large number of LC peaks remain not identifiable .
2) MS/MS library limitations• MS/MS spectral search is usually manually performed and time-
consuming; • Automated scoring for spectral matching is usually not available;• The fragmentation needs to be manually interpreted, which is not
only time-consuming but also prone to errors.
3) There is no good solution for unknowns.
The Orbitrap Fusion MS enables fast parallel data acquisition on a UHPLC timescale with a variety of dissociation choices The Orbitrap Fusion MS o ers ultimate flexibility in fragmentation methods at all stages of MSn analysis, with fragment ions detected by either mass analyzer. CID-HCD, HCD-CID, and HCD-HCD can all be set up as simple as the conventional CID-CID. This complete flexibility facilitates the examination of fragmentation pathways that leads to the most comprehensive structural information.
mzCloud MSn library enables MSn search for both high and low resolution mzCloud is a multistage MS library. Based on the spectral tree similarity, an algorithm called precursor ion fingerprinting (PIF) enables the identification of compounds even if they are not in the library, and the fragmental peaks can be annotated [2]. Powered by the novel MS and the mzCloud library, an improved, more connected workflow is shown below:
Major benefits of the improved workflow: 1) HCD MS/MS can be performed simultaneously with full scan MS on the
UHPLC timescale with ion trap detection at >22 Hz.2) Simultaneous ddMSn ion tree with flexible combinations of CID and/or HCD
fragmentation with ion trap and/or OrbitrapTM detection. 2) Both the MS2 and MSn will be automatically searched in mzCloud, the
matching entry will be scored based on PIF, and the fragments will be annotated.
4) De novo structural identification is enabled by using this workflow.
OverviewPurpose: To demonstrate the capability of the Thermo Scientific™ Orbitrap Fusion™ Tribrid™ Mass Spectrometer in combination with MSn library for metabolite structural identification.
Methods: UHPLC coupled to the Orbitrap Fusion MS to simultaneously perform global profiling and structural elucidation with MSn.
Results: Within UHPLC timescale (2-3 second FWHM), >10 cycles can be finished with fast ion trap HCD MS2 for top 11 precursors, each with ion trees of MS2-MS3(top2)-MS4(top2).
IntroductionThe structural identification of metabolites represents a significant challenge in metabolomics study. Multistage mass spectrometry (MSn) is a powerful tool for compound identification and structural elucidation that goes beyond identifying the exact same compounds, to discovering additional compounds with structural homology to those in the library. Combining with high resolution accurate mass (HRAM) measurement, MSn is highly e ective in identifying the unknown but biologically relevant compounds in metabolomics studies[1]. However, the speed on current platforms is yet to be e ectively compatible with UHPLC front end, which limited such application in biological samples.
Presented here is a new platform to identify endogenous metabolites using a novel high performance Orbitrap hybrid instrument in conjunction with UHPLC and an MSn library.
Methods Sample Preparation
Urine samples from an adult male were analyzed. The urine samples were diluted 1 to 10 with water and processed with a 3kD MWCO filter.
For plasma samples, proteins were removed with cold methanol and centrifugation, followed by centrifugal evaporation at 35 oC. The residue was reconstituted in MeOH/Water, 1/9, and also processed with a 3kD MWCO filter.
Liquid ChromatographyUHPLC separation was implemented on a Dionex Ultimate 3000 HPG (high-pressure gradient) pump using Hypersil GOLD RP C18 column at 450 L/min, column temperature at 55 oC. LC solvents were 0.1% FA (A) and 0.1% FA in MeOH (B). A linear gradient was applied from 0.5-50% B for 5.5 min, followed by increasing to 98% at 6 min, held 98% B for 6 min, then decreased to 0.5% at 13min, and equilibrated for another 2 min.
FIGURE 1. Schematic of Orbitrap Fusion Tribrid Mass Spectrometer
A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzCloud Library Junhua Wang1, David A. Peake1, Robert Mistrik2, Yingying Huang11Thermo Fisher Scientific Inc, San Jose, CA; 2HighChem, Ltd., Bratislava, Slovakia
Results
S-Lens
Active beam guide (ABG)
Ultra-high-field Orbitrap mass analyzer
Large-surface-Area detector (LSAD)
Ion-routing multipole
Dual-pressure linear ion trap
Quadrupole mass filter
C-Trap
EASY-Internal Calibration ion source
FIGURE 2. Ultrahigh resolution resolves the isotopes
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
PositiveMode20130518092831 #654 RT: 2.60 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #539 RT: 1.44 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #511 RT: 1.33 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #615 RT: 1.97 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
PositiveMode20130518092831 #237 RT: 0.51 AV: 1 NL:T: FTMS + p ESI Full ms [100.00-212.08]
151.05 151.06m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R=30k
R=60k
R=120k! =240kR=500k
L-Methionine C5H11NO2S
0.7ppm
Fine isotope pattern
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
PositiveSimulated
R=500,000
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
151.0617C4
13CH12O2NS
151.0625C5H12O17ONS
151.0646C5
13C14CH918O17O
151.0577C5H12O2N33S
151.55414C4H10
18O233S
PositiveObserved
Negative20130518111518 #1449 RT: 8.07 AV: 1 NL: 1.00E6T: FTMS - p ESI Full ms [140.00-220.00]
179 180 181 182m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0576R=550402
180.0611R=330000
NegativeFull Scan
0.55 ppm
Negative20130518111518 #158 RT: 1.66 AV: 1 NL: 1.24E6T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0573R=67606
Negative20130518111518 #110 RT: 1.34 AV: 1 NL: 8.48E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=148204
179.0558R=130000
Negative20130518111518 #54 RT: 0.83 AV: 1 NL: 9.54E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0574R=304604
179.0560R=275000
Negative20130518111518 #21 RT: 0.40 AV: 1 NL: 7.38E5T: FTMS - p ESI Full ms [140.00-220.00]
179.050 179.055 179.060 179.065m/z
0
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=500,000200 M
1 M
1.1 ppm
0.56 ppm
0.55 ppm
0.55 ppm
0.56 ppm
1.7 ppm
0.55 ppm
FIGURE 3. Ultrahigh resolution resolves isobaric metabolites
RT: 2.75160 - 2.86730
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
NL:3.23E7Base Peak m/z= 205.09525-205.09935 F: ms MS JWUrine_FS240k_LM_Re
NL:2.85E7Base Peak m/z= 205.09525-205.09935 MS jwurine_fs240k_nolm_re
PBUrine_MS2_120k_600ms_bothLM #824 RT: 2.82 AV: 1 NL: 8.68E6F: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
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 188.07079170.06030without Int. Cal.Avg =1.5 ppm
Full Scan MS HCD MS/MSJWUrine_MS2_120k_600ms_bothLM #896 RT: 2.84 AV: 1 NL: 1.08E7T: FTMS + p ESI d Full ms2 [email protected] [50.00-216.00]
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
146.06020
118.06530
132.08090 170.06015 188.0706391.05422
0.2 ppmO
NH
NH
OHO
NH
0.9 ppm 0.7 ppmNH •
0.4 ppm
0.8 ppm
N2H
1 ppm
NH
N 2HNH
-0.1 ppm
with Int. Cal.Avg = 0.4 ppm
1.1 ppm
1.8 ppm
2.0 ppm
1.7 ppm
1.9ppm
2.9 ppm
1.1 ppm
with Int. Cal.
without Int. Cal.
Metabolite Identification Requires High Resolution and Isotope Ratio Measurements Structure elucidation of unknown small molecules by mass spectrometry is a great challenge. The first crucial step is to obtain correct elemental compositions.
Natural occurring elements can be monoisotopic (F, Na, P, I) or polyisotopic (H, C, N, O, S, Cl, Br) [1]. The molecule L-Methionine C5H11NO2S contains multiple polyisotopic elements, especially the S, which result in multiple isotopic peaks needing resolving power above 240,000 at m/z 200 (FWHM). Figure 2A shows the better isotope pattern of molecular ion [M+1+H]+. With increasing resolution up to 450,000 (FWHM), all five isotope peaks are clearly resolved. Figure 2B shows the simulated isotope pattern.
A
B
Metabolite Identification and Quantitation in Complex Samples Requires High ResolutionD-Glucose C6H12O6 and Paraxanthine C7H8N4O2 co-exist in human blood at distinct concentration levels: 5300 M (D-Glucose) vs. 10 M (Paraxanthine) [3]. Although D-Glucose is hundreds of fold more concentrated, it is less readily detectable because of its low ionization e ciency. Their masses di erentiate by 0.0013 amu (179.0561 vs. 179.0574), making the detection of these individual components very challenging. Figure 3 shows that, with >300,000 resolving power, the 200 M (D-Glucose) and 1 M (Paraxanthine) can be readily separated and quantified.
FIGURE 4. Easy Internal Calibration
Metabolite Identification Requires High Accuracy of Mass Measurement in Both MS Full Scan Level and MS/MS LevelFor an unknown metabolite, the number of calculated chemically possible formulae could be hundreds (20 ppm tolerance window), but the possibilities significantly reduce to tens (3 ppm) and several if the tolerance window is narrowed to sub-ppm. However, due to the large amount of known isomeric structures, one single formula may correspond to hundreds of structural isomers, thus structure identification of metabolite isomer using only MS full scan is not feasible. MS/MS and MSn techniques can help solve this analytical problem.
The Orbitrap Fusion MS has a built-in internal calibration functionality (Easy IC). Figure 4 shows LC-MS/MS data of L-Tryptophan from an urine sample with and without internal calibration. Sub-ppm mass accuracy was observed for MS and MS2 fragments with IC activated.
FIGURE 5. Rapid Ion Tree Acquisition on UHPLC Timescale from a Urine Sample (1 MS, 2 MS2 and 4 MS3 in 0.6sec/cycle).(A) Ion trap HCD MS2 followed by Orbitrap CID MS3 were acquired for the precursor ion m/z 181.0721. (B) Spectral tree search in mzCloud matches the MS3 with MS3 from theophylline with the highest score, indicating the unknown must have the same substructure as theophylline.
ConclusionThe Orbitrap Fusion MS o ers ultra high resolution and high fidelity in isotope ratio measurements of metabolites. This allows metabolites in complex mixture with a wide dynamic range be resolved and detected. The Easy-Internal Calibration allows compound assignment be readily done with confidence. The Orbitrap Fusion MS provides ultimate flexibility that facilitates the examination of multiple structural pathways for more structural information at higher speed.
The mzCloud library allows metabolite identification with MS2 and/or MSn data in an automated fashion. The identification based on multistage MS spectral trees and PIF algorithm opens the possibility to identify unknown metabolites and isomers. An improved metabolomics workflow is enabled.
References1. Budzikiewicz H, Grigsby RD, Mass Spectrom. Rev. 2006, 25(1):146-157.
2. Sheldon MT, Mistrik R, Croley TR, J. Am. Soc. Mass Spectrom. 2009, 20(3):370-376
3. http://www.hmdb.ca/metabolites/HMDB00122 & HMDB01860
A
BTheophylline
MS3
isomers
Query (down) and library spectra (up) matchScore
N
NH NH
N
NH
1.5 ppm 1.5 ppm
OH
NH
1 ppm
2.1 ppm
FIGURE 1. The Orbitrap Fusion MS and the Drag and Drop Instrument Method Editor.
O
N
NH NH
ASMS13_MP 045_JWang_E 07/16S
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