data acquisition and analysis in mass spectrometry based metabolomics
DESCRIPTION
Data Acquisition and Analysis in Mass Spectrometry Based Metabolomics. Pavel Aronov BioCyc workshop October 27, 2010. Outline. Fundamentals of Mass Spectrometry Data Acquisition and Analysis in GC-MS based Metabolomics Data Acquisition and Analysis in LC-MS based Metabolomics. - PowerPoint PPT PresentationTRANSCRIPT
Data Acquisition and Data Acquisition and Analysis in Mass Analysis in Mass
Spectrometry Based Spectrometry Based MetabolomicsMetabolomics
Pavel AronovPavel Aronov
BioCyc workshopBioCyc workshop
October 27, 2010October 27, 2010
OutlineOutline
Fundamentals of Mass SpectrometryFundamentals of Mass Spectrometry
Data Acquisition and Analysis in GC-Data Acquisition and Analysis in GC-MS based MetabolomicsMS based Metabolomics
Data Acquisition and Analysis in LC-Data Acquisition and Analysis in LC-MS based MetabolomicsMS based Metabolomics
How to analyze tryptophan or any How to analyze tryptophan or any other metabolite?other metabolite?
Two most common techniques in analytical Two most common techniques in analytical chemistry to determine or confirm chemical chemistry to determine or confirm chemical structure:structure:
Nuclear Magnetic Resonance/NMR (1940s, Felix Nuclear Magnetic Resonance/NMR (1940s, Felix Bloch at Stanford University)Bloch at Stanford University)Excellent structural informationExcellent structural information
Mass Spectrometry (1900s, JJ Thompson at Mass Spectrometry (1900s, JJ Thompson at Cambridge University)Cambridge University)Excellent sensitivityExcellent sensitivity
What is a mass spectrometer?What is a mass spectrometer?
Ion Source Mass Analyzer Detector
AtmosphereVacuum
M
M+
M+
M
M
M+
M+
Mass Spectrometer
Measured value: mass-to-charge ratio M/Z
Mass UnitsMass Units
Unit of mass:Unit of mass:1/12 mass of carbon-12 atom 1/12 mass of carbon-12 atom 1 u or 1 Da1 u or 1 Da
Unit of mass-to-chargeUnit of mass-to-charge1 Da / z = 1 Th (Thompson)1 Da / z = 1 Th (Thompson)m/z 205m/z 205
For metabolites usually z = 1, For metabolites usually z = 1, Hence 1 Da is equivalent to 1 ThHence 1 Da is equivalent to 1 Th
Monoisotopic vs Average Mass Monoisotopic vs Average Mass
Two stable isotopes important in biochemistryTwo stable isotopes important in biochemistryCarbon-12 (100 %) and Carbon-13 (~1.1 %)Carbon-12 (100 %) and Carbon-13 (~1.1 %)Sulfur-32 (100 %) and Sulfur-34 (4.4 %)Sulfur-32 (100 %) and Sulfur-34 (4.4 %)
Tryptophan statistically can contain:Tryptophan statistically can contain:nono carbon-12 (M): 204.09 Da (100 %) carbon-12 (M): 204.09 Da (100 %) one carbon-13 (M+1): 205.09 Da (11.9 %)one carbon-13 (M+1): 205.09 Da (11.9 %)two carbons-13 (M+2): 206.09 Da (1.4 %)two carbons-13 (M+2): 206.09 Da (1.4 %)
These are These are monoisotopicmonoisotopic masses masses
Average mass = (204.09 *100 + 205.09*11.9 + Average mass = (204.09 *100 + 205.09*11.9 + 206.09*1.4)/113.2 = 204.22 (molecular 206.09*1.4)/113.2 = 204.22 (molecular weight, g/mol)weight, g/mol)
O
OH
NH2HN
C11H12N2O2
Fall 0022 DS
mass203 204 205 206
%
0
100
vitD051608sample001 (0.005) Is (1.00,1.00) C11H12N2O28.73e12204.09
205.09
206.10
Mass defectMass defect
1H (p+e-) 1.0078 u12C 12.0000 u14N 14.0031 u16O 15.9949 un 1.0087 u
Carbon-12: 6 protons, 6 neutron and 6 electrons6 x 1.0078 u + 6 x 1.0087 u = 12.0990 u
Mass Defect = 12.0990 u – 12.0000 u = 0.0990 u
E = mc2
0.1 u = 93 MeV
Elemental composition from Elemental composition from accurate massaccurate mass
1H 1.0078 u12C 12.0000 u14N 14.0031 u16O 15.9949 u
What is 28 u? N2 (2 x 14 u), CO (12 u + 16 u) or C2H4 (2 x 12 u + 4 x 1 u)?
What is 28.0313 u? [high accuracy]C2H4 (2 x 12.0000 u + 4 x 1.0078 u)
High resolution mass High resolution mass spectrometryspectrometry
99m/z
560 561 562 563 564
%
0
100
%
0
100562.19
561.18
563.20
564.20
561.14
562.10
563.06
0.06 amuFWHM
0.8 amuFWHM
High Resolution: R = 561/0.06 ~ 9,000
TOF: 7,000-50,000Orbitrap: 104-105
FT ICR: 105-106
Nominal Mass Resolution (<1000)R = 561/0.8 ~ 700
Quadrupoles and ion traps, some TOFs
Two types of ions in mass spectrometry:
Odd Electron (OE) Ions Typically generated by electron ionization (GC/MS):
Even Electron (EE) IonsTypically generated by chemical ionization techniques and electrospray
Mass of an electron becomes Mass of an electron becomes important at high accuraciesimportant at high accuracies
C11H12N2O2C11H12N2O2
204.08988 Da (2.6 ppm error)
204.08933 Da(true mass)
C11H12N2O2 C11H13N2O2
C11H13N2O2
205. 09715 Da (true mass)
205. 09770 Da (2.6 ppm error)
e
0.00055 Da
Modern instruments can achieve < 1 ppm accuracy
Identification based on accurate Identification based on accurate massmass
211 212 213 214 215m/z
0
10
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30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
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100
Re
lativ
e A
bu
nd
an
ce
212.00217C 8 H6 O4 N S-0.63646 ppm
212.00230C 8 H6 O4 N S0.00000 ppm
NL:6.95E6MeyerT_100422_sample0062#636 RT: 6.29 AV: 1 T: FTMS {1,1} - p ESI Full ms [70.00-800.00]
NL:8.59E5
C 8 H6 O4 N S: C 8 H6 O4 N1 S 1pa Chrg -1
Theoretical spectrum
Acquired spectrum
Ma
tch
ing
ac
cu
rate
mas
sa
nd
iso
top
ic p
eak
ra
tio
HN
OSO3
Error = -0.00013 Da/212.0023 Da * 1000,000 = 0.6 parts per million (ppm)
Confirmation of structure from Confirmation of structure from isotopes (M+2)isotopes (M+2)
Theoretical spectrum
Acquired spectrum
Ma
tch
ing
ac
cu
rate
mas
sa
nd
iso
top
ic p
eak
ra
tio
HN
OSO3
213.94 213.96 213.98 214.00 214.02 214.04 214.06 214.08m/z
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100R
ela
tive
Ab
un
da
nce
213.99796
213.99810
NL:3.24E5MeyerT_100422_sample0062#636 RT: 6.29 AV: 1 T: FTMS {1,1} - p ESI Full ms [70.00-800.00]
NL:3.88E4
C 8 H6 O4 N S: C 8 H6 O4 N1 S 1pa Chrg -1
Tandem Mass SpectrometryTandem Mass Spectrometry
Ion Source
Mass Analyzer
1Detector
AtmosphereVacuum
M
M+
M+
M
M
F+
F+
Mass Spectrometer
HPLC
Mass Analyzer
2
Collision Cell
F+
F+
M+
M+
MS/MS of isomersMS/MS of isomers
Prostaglandin A1336.2301 amu
Prostaglandin B1336.2301 amu
ChromatographyChromatography
Time6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00
%
0
100)
14.40
11.82
10.43
9.00
16.73
18.84
30.0520.77
28.4422.5327.0724.16
25.66
C8C9
C10C12
C14C16
C18C20
C22
C30
Separation by volatility and polarity (gas chromatography/GC)or polarity (liquid chromatography/LC)
Gas chromatography ofhydrocarbons
2D dimensionality of metabolomics data2D dimensionality of metabolomics datain LC-MS and GC-MSin LC-MS and GC-MS
GC-MS and LC-MSGC-MS and LC-MS
LCGC
-Derivatization usually required (except VOC)-Upper mass limit at ~400-500 amu-Preferred for small polar metabolites (primary metabolism)-Relatively high peak capacity
-No derivatization usually required -Upper mass is limited by column permeability -Preferred for bigger molecules (e.g. some lipids, secondary metabolites) -Relatively low peak capacity
-EI ion source (extensive fragmentation, reproducible, libraries available
-CI ion source (little fragmentation, advantage for accurate mass measurement
-ESI ion source (ionic compounds, ion suppression)
-APCI ion source (less ion suppression and more amenable for non polar compounds than ESI but usually lower sensitivity)
MS
Types of Experiments in Types of Experiments in MetabolomicsMetabolomics
targeted non-targeted
• Number of analyzed metabolites is limited by the number of available standards
• Absolute quantitation of metabolites (nM, mg/mL)
• Selective MS detectors (quadrupoles, triple quadrupoles)
•Number of analyzed metabolites is limited by capacity of analytical instrumentation
• Relative quantitation of metabolites (fold)
•Scanning MS detectors (ion trap, TOF, FT)
Bottlenecks in MetabolomicsBottlenecks in Metabolomics
1-Identification of metabolites; 35%
2-Assigning biological significance; 22%3-Data processing/reduction; 14%
4-Sample preparation; 8%
5-No opinion; 6%
6-Statistical analysis; 5%
7-Validation/Utility Studies; 5%8-Data acquisition/throughput; 3%
9-Other; 2%ASMS09 survey: metabolomics bottlenecks
throughput (3 %) vs. post-acquisition bottlenecks (5 + 35 + 22 + 14 = 76 %)
GC-MS based metabolomics: GC-MS based metabolomics: overviewoverview
50 - 50 - 600 (400)600 (400) amu mass range amu mass range mono- and disaccharides, amino acids, fatty acids (mostly mono- and disaccharides, amino acids, fatty acids (mostly
primary metabolites)primary metabolites)
Derivatization usually required Derivatization usually required
GC-MS: derivatizationGC-MS: derivatization
40 mg/mL in pyridine at 37˚C for 90 min40 mg/mL in pyridine at 37˚C for 90 min Prevents Prevents αα-ketoacids from thermal decarboxylation-ketoacids from thermal decarboxylation Keeps sugars in open conformation to minimize Keeps sugars in open conformation to minimize
number of conformation and relieve steric hindrences number of conformation and relieve steric hindrences for next stepfor next step
OH
OHHO
OHO OH
O
OHHO
OHHO OH
N
OHHO
OHHO OH
OCH3
H3CO
NH2
α/β epimers
Syn/anti isomers
GC-MS: derivatizationGC-MS: derivatization
MSTFA, 1% TMCS at 50˚C for 30 minMSTFA, 1% TMCS at 50˚C for 30 min Substitution of active hydrogensSubstitution of active hydrogens Incomplete derivatization possibleIncomplete derivatization possible
O
HO
NH2 O
O
HN
Si
Si
O
O
N
Si
Si
Si
MSTFA MSTFA
GC-MS data analysisGC-MS data analysisSH-15 H
Time10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 47.50 50.00
%
0
100
G-603-SH-15-H Scan EI+ TIC
1.34e634.5522.8314.24
13.079.40
9.8710.03
12.44
13.69
14.48 21.25
15.00
15.06
16.23
16.10 18.05
16.9018.37 20.99
20.0119.11
22.55
21.78
23.34
27.1523.4225.42
23.98
26.66
26.09
28.83
27.31
28.22
30.66
30.07
32.95
30.81
31.32
32.04
34.39
33.47
36.58
35.50
49.65
40.57
39.9237.3837.62
38.49 42.5841.9242.80
Electron Ionization in GC-MS Electron Ionization in GC-MS
70 eV >> energy of chemical 70 eV >> energy of chemical bondbond
Highly reproducibleHighly reproducible Extensive fragmentationExtensive fragmentation Often no molecular ion observedOften no molecular ion observed
EI: alpha-cleavage [EI: alpha-cleavage [aa ] more common ] more common
CID MS/MS: inductive cleavage [CID MS/MS: inductive cleavage [ii ] common ] common
OH OHaOH2 i
GC-MS: present and futureGC-MS: present and futureCurrent GC-MS metabolomics platforms use:1) nominal resolution mass analyzers (no accurate mass and elemental composition) 2) electron ionization ion sourceOE molecular ions, extensive fragmentation, often molecular ion is not observed
Advantages:1) Low cost2) Good chromatographic separation for many small polar metabolites after derivatization3) Extensive libraries of fragmentation spectra help identification4) Retention time is to some extent predictable (retention indices)
Trends:1) Development of high resolution instruments for GC/MS2) Development of soft ionization sources similar to LC/MS(EE ions, no fragments)
GC-MS data analysisGC-MS data analysis Deconvolution of mass spectra based on Deconvolution of mass spectra based on
chromatographic profiles (e.g freeware chromatographic profiles (e.g freeware AMDIS)AMDIS)
Identification of metabolites based on Identification of metabolites based on matching to spectral libraries and matching to spectral libraries and retention indicesretention indices
Automated processing routines exist for Automated processing routines exist for some GC-MS instrument (SetupX and some GC-MS instrument (SetupX and BinBase)BinBase)
Application ExamplesApplication Examples
- listeria 1st inj
Time6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50
%
0
100
6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50
%
0
100
ARONOVP_100819_SAMPLE004_STER Scan EI+ TIC
2.31e7
6.16
ARONOVP_100819_SAMPLE005_LIST Scan EI+ TIC
2.31e710.67
6.16
- cells
+ cells
Glycine-2TMS
Application Examples: AMDISApplication Examples: AMDIS
Peak of interest
Acquired mass spectrum
Library mass spectrum(glycine-2TMS)
LC-MS based metabolomicsLC-MS based metabolomics
Combination of ionization modes is preferred (ESI, Combination of ionization modes is preferred (ESI, APCI, +, -)APCI, +, -)
Reversed phase LC for non-polar metabolites and Reversed phase LC for non-polar metabolites and hydrophilic interaction chromatography (HILIC) for hydrophilic interaction chromatography (HILIC) for polar metabolitespolar metabolites
Detection of spectral “features” (ions) using Detection of spectral “features” (ions) using metabolomics softwaremetabolomics software
Identification based on accurate mass, and Identification based on accurate mass, and fragmentation (MS/MS libraries)fragmentation (MS/MS libraries)
Electrospray Ionization (ESI)Electrospray Ionization (ESI)
R + H+
R – H+
[R+H]+
[R – H]+
Positive ESI
Negative ESI
APCIAPCI ESIESISoft ionization, pseudomolecular ions [M + H]Soft ionization, pseudomolecular ions [M + H]++, [M - H], [M - H]- - ,[M + Na],[M + Na]++, [M , [M + Cl]+ Cl]--
Volatile mobile phase, no inorganic salts (phosphate buffer)Volatile mobile phase, no inorganic salts (phosphate buffer)
Ionization in gas phaseIonization in gas phase Ionization in liquid phaseIonization in liquid phase
High ionization efficiency for High ionization efficiency for compounds with high proton compounds with high proton affinity in gas phaseaffinity in gas phase
High ionization efficiency for High ionization efficiency for compound ionic in a solutioncompound ionic in a solution
Usually singly charged ionsUsually singly charged ions Multiply charged ions common for Multiply charged ions common for large biomolecules (proteins, large biomolecules (proteins, nucleic acids)nucleic acids)
Compatible with reverse and Compatible with reverse and normal phase, normal phase,
Reverse phase, Reverse phase, Mobile phase must be conductiveMobile phase must be conductive
Ion suppression commonIon suppression common
Combination of Acquisition ModesCombination of Acquisition Modes
Separation modes: Reversed phase and HILIC
Ionization modes: ESI and APCI or combined ESI/APCI (MM)
Ionization polarities: + and -
Nordstrom A. et al, Anal Chem, 2008.
RP and HILIC liquid chromatographyRP and HILIC liquid chromatography
RT: 0.00 - 10.03
0 1 2 3 4 5 6 7 8 9 10Time (min)
0
5
10
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Re
lative
Ab
un
da
nce
1.63496.34
2.57114.07
2.55114.07
1.42758.57
2.53114.07
4.38269.004.14
269.00 4.54269.00
4.03269.00
2.93144.10 4.77
104.99 7.60233.24 7.90
233.247.45
233.245.93
255.231.22
288.29 8.45233.243.04
118.091.77496.34
8.87233.240.92
332.339.33
233.24
0.86233.25
NL:1.72E8TIC MS MeyerT_100127_sample067
RT: 0.00 - 10.00
0 1 2 3 4 5 6 7 8 9 10Time (min)
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10
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Re
lative
Ab
un
da
nce
1.12114.07
2.31166.09
1.69132.10
3.29188.07
4.03268.15
2.45232.03
3.93102.09
6.6974.10
7.1274.10
4.52102.09
2.80102.09
7.6674.10
5.2174.10
5.4374.10
8.9674.10
8.4674.10 9.02
74.100.11
74.10
NL:8.02E7TIC MS MeyerT_100127_sample034
Reversed Phase C18
Aminopropyl HILICBetter retention for polar molecules
NH2
N
NO
Creatinine
Creatinine
LC-MS: Data AnalysisLC-MS: Data Analysis Alignment of chromatograms (optional)Alignment of chromatograms (optional)
Detection of ‘features’ in mass chromatogramsDetection of ‘features’ in mass chromatograms
Removal of isotopic peaks, adducts, fragments etc to Removal of isotopic peaks, adducts, fragments etc to improve statisticsimprove statistics
Statistical analysisStatistical analysis
Identification based on accurate mass, MS/MS spectra Identification based on accurate mass, MS/MS spectra and comparison with standardsand comparison with standards
Example:Example:Search for bacterial metabolites in humans Search for bacterial metabolites in humans comparing two groups: controls and people comparing two groups: controls and people who underwent colectomy (no colon bacteria) who underwent colectomy (no colon bacteria)
Initially software detected 900 features in positive ESI mode
After features with missing chromatographic profile were removed769 features left (visual inspection)
After isotopes were removed, 554 features left. Only at this point,these are likely molecular ions of individual metabolites
AdductsAdductsMeyerT_100422_sample0088 4/27/2010 7:23:42 PMC18 pos R5
RT: 14.99 - 16.15
15.0 15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9 16.0 16.1
Time (min)
0
50
1000
50
100
Rel
ativ
e A
bund
ance
0
50
10015.59 15.61
15.83 15.86 15.97 16.01 16.0615.3415.59
16.03
16.1215.09 15.9015.12 15.1815.61
15.78 15.84 15.97 16.08
NL:1.53E6
m/z= 398.95-399.26 MS MeyerT_100422_sample0088
NL:1.37E6
m/z= 416.11-416.28 MS MeyerT_100422_sample0088
NL:6.43E5
m/z= 421.05-421.26 MS MeyerT_100422_sample0088
MeyerT_100422_sample0088 #1581-1600 RT: 15.51-15.69 AV: 20 NL: 7.32E5T: FTMS {1,1} + p ESI Full ms [70.00-800.00]
398 400 402 404 406 408 410 412 414 416 418 420 422 424 426
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
399.1856
416.2120
421.1673
400.1888 417.2153
422.1704401.1905 418.2171 423.1722413.2656407.1887403.0866397.1851
420.3664
409.9785415.2011
M + H M + NH4
M + Na
M + H
M + NH4
M + Na
Fragments in LC-MSFragments in LC-MSMeyerT_100422_sample0088 4/27/2010 7:23:42 PMC18 pos R5
RT: 6.69 - 8.06
6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0
Time (min)
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance
7.317.337.29
7.35
7.39
7.51 7.55 7.60 7.66 7.74 7.80 7.86 7.946.876.78 7.997.176.73 7.086.91 7.00
7.317.337.29
7.35
7.39
7.41
7.51 7.55 7.60 7.66 8.007.17 7.766.78 7.937.066.73 6.92 6.98 7.12 7.806.85
NL:2.23E5
m/z= 118.05-118.09 MS MeyerT_100422_sample0088
NL:2.38E5
m/z= 118.05-118.09 MS MeyerT_100422_sample0088
MeyerT_100422_sample0088 #740-782 RT: 7.16-7.52 AV: 43 NL: 3.04E6T: FTMS {1,1} + p ESI Full ms [70.00-800.00]
115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
180.0651
181.0684122.5471
162.0547118.0651 182.9847154.9899 167.0125134.0599 149.0231 195.0873173.0298141.9584125.9862
176.9715
Hyppuric acid
Hyppuric acid
C8H8N – indole?No, fragment of hyppuric acidNot confirmed by GC-MS either
m/z 118.0651
Identification toolsIdentification tools
Accurate mass search (BioCyc, Accurate mass search (BioCyc, HMDB, Metlin)HMDB, Metlin)
MS/MS search (Metlin, MassBank)MS/MS search (Metlin, MassBank)
In addition, many MS manufacturers In addition, many MS manufacturers offer proprietary tools for structure offer proprietary tools for structure elucidationelucidation
MassBank MS/MSMassBank MS/MS
sulfate
m/z 132C8H6NO
LC-MS Data Analysis SummaryLC-MS Data Analysis Summary
Not every peak detected by a mass spectrometer Not every peak detected by a mass spectrometer represents an individual metaboliterepresents an individual metabolite
Automated data processing helps to reduce the Automated data processing helps to reduce the amount of routine work, however human amount of routine work, however human intervention is still required intervention is still required
Accurate mass measurements and MS/MS allow Accurate mass measurements and MS/MS allow to determine elemental composition of unknowns to determine elemental composition of unknowns and their structural components. Confirmation and their structural components. Confirmation with chemical standards is still required with chemical standards is still required