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

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

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)

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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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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

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100

Rel

ativ

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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