Improving the Metabolomics Mass Spectrometry Workflow ... · •Most systems use either liquid or...

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Metabolite Extraction and Platforms for Metabolomic Studies

Andrew D. Patterson, PhDAssociate Professor of Molecular ToxicologyPenn State [email protected]

Resources

• Metabolomics Workbench – www.metabolomicsworkbench.org– Large resource of experimental protocols, datasets, and other resources

• XCMS Institute– https://xcmsonline.scripps.edu/institute– Great tutorials on chromatography, platforms, databases

• Metabolomics Society Forums– http://www.metabolomics-forum.com/

• Twitter – #metabolomics

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Objectives

At the conclusion of this lesson, students will be able to:

• Define factors that influence metabolite extraction and describe their impact on metabolomic studies

• Explain the value of orthogonal approaches for improved metabolite identification and quantitation

Which parts of the metabolomic process might influence your data?

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Other Classes of Metabolites

• Where might you have trouble extracting everything from a particular class of metabolites?

• Example: Are all bile acids the same in terms of general solubility in aqueous or organic solvents?

Matrix Effects

• Challenges with urine–

–

• Challenges with blood, serum, or plasma–

–

• Challenges with tissue–

–

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Extraction of Metabolites

Nucleic Acid Extraction

What do we know about our target analyte?

• Negatively charged phosphate backbone (polar)• Need to remove proteins, lipids, etc

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About Solvents

OH

PHENOL

1.07 g/cm3

O

H H

WATER

1.00 g/cm3

Cl

Cl

HCl

CHLOROFORM

1.49 g/cm3

POLARITY

Protein Chemistry

https://en.wikipedia.org/wiki/Proteinogenic_amino_acid

Denature proteins

• Hydrophobic amino acids face phenol:chloroform

• Phe, Tyr, Leu

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Liquid Liquid Extraction

DNA or RNA(aqueous)

Protein

Lipids (phenol:chloroform)

DNA Extraction Protocol

• Disrupt tissue in phenol:chloroform

**chloroform prevents small amounts of water in phenol from dissolving mRNA**adjust pH to favor DNA (basic) or RNA (acidic) isolation

• Centrifuge to separate layers• Dehydrate with alcohol

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What could go wrong?

• Solvents not appropriate or prepared incorrectly

– pH incorrect

– Ratios incorrect

• Contamination of solvents or buffers

• What else can you think of?

Standard Operating Procedures

• Saves time and prevents mistakes

• Consistent results

• Checking in samples (sample lists, location)

• Labeling and storing samples (aliquot)

• Metabolite extraction (targeted or global)

• Acquiring data on various platforms (MS, NMR)

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Even with SOPs…

Note influence of individual preparing pooled samples. Easy to see who prepared what.

DECAF COFFEE

SUMATRACOFFEE

COLUMBIAN COFFEE

Common Solvents for Metabolomics

• Methanol

• Acetonitrile

• Chloroform

• Methyl tert-butyl ether (MTBE)

• Water

NH3C

CH3HO

Cl

Cl

HCl

H3C

H3C

CH3

CH3O

O

H

H

List is not inclusive

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Methanol

• Relatively inexpensive compared to acetonitrile

• Not regulated like ethanol

• Easy to evaporate

• Extracts polar and (some) non-polar molecules – why?

Acetonitrile

• Advantages mostly for chromatography– Reduced absorbance for UV based methods

– Reduced pressure compared to methanol

– Greater elution strength (generally)

– HILIC applications

• Expensive– Isolated as a byproduct not produced directly

– Shortages can influence price and availability

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Chloroform vs MTBE

• Chloroform densitiy – 1.49 g/cm3

• MTBE density – 0.740 g/cm3

• Toxicity of chloroform (check out ATSDR.CDC.GOV)

• Still made need to tailor specific extractions for lipid classes (hexane for TAGs or MTBE for Cer)

– More detail checkout cyberlipid.org or lipidmaps.org

SO WHAT?

Metabolomics

Metabolomics is the systematic analysis of the unique chemical fingerprints left behind by specific cellular processes

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FOOD

PESTICIDES

COSMETICS

POLLUTANTS

DRUGS

XEN

OB

IOTI

C

MET

AB

OLI

SM

END

OG

ENO

US

MET

AB

OLI

SM

METABOLOME

GUT MICROBIOTA

Metabolomics

All “-omics” based scientific disciplines aim at the collectivecharacterization and measurement of their particularconstituent molecules

• A comprehensive approach to study complete pools ofbiological molecules

• Defines the structure, function and dynamics of anorganism

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Metabolomics

Vast chemical diversity among small molecule metaboliteshas made extended coverage of the metabolomechallenging

• Size (50 – 1500 Da)

• Concentration ( pM – mM)

• Physicochemical properties (diverse log P values)

• Stereochemistry (distinct biological activity)

EHP 2014 Vol 122 Number 8http://ehp.niehs.nih.gov/1308015/

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http://journal.frontiersin.org/article/10.3389/fimmu.2016.00044/full

Metabolite Extraction

• Currently no analytical technique exists that is capable ofmeasurement of all classes of cellular metabolites

• Metabolite extraction is a crucial step in any metabolomics study

– Critical to both targeted and global based profiling strategies

• Optimized extraction methodology should fulfill several criteria:

– Extract the largest number of metabolites

– Unbiased and non-selective - physical or chemical propertiesof a molecule

– Non-destructive - no modification of metabolites

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organic phase

aqueous phase

KD

HAorg

HAaq+ H20 H30+ + A-Ka

organic phase

aqueous phase

KD

HAorg

HAaq

Separation of Metabolites

• Mass spectrometry usually requires some form of chromatographic separation

• Most systems use either liquid or gas chromatography

• Fractionation of sample components simplifies the resulting mass spectra while ensuring more accurate compound identification

• Capacity factor (k) is critical to optimizing resolution

• Increased resolution allows longer MS dwell times resulting in better signal/noise ratios

• Inadequate chromatographic separation of metabolites results in:

• signal suppression – ion suppression

• compromised metabolite quantification

• reduced metabolite coverage

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Ceramide Physicochemical Properties

• Ceramides are a family of waxy lipid molecules.– Name derived from the latin word: cera = waxy + amide

• Ceramides are comprised of: – sphingosine: 18 carbon unsaturated amino alcohol – fatty acid moiety – amide bond

• Ceramides are not water soluble:– Very hydrophobic– Confined to cellular membranes– Participate in lipid raft formation– >200 structurally distinct species have been identified in

mammalian cells

Ceramide General Structure

• Ceramide (d18:1/16:0)

• 2-amino-1,3-octadec-4-ene-diol

• Amino alcohol (sphingoid) backbone

• Palmitic acid

• Fatty acyl group

• Ceramide (d18:1/24:1(15Z))

• 2-amino-1,3-octadec-4-ene-diol• Amino alcohol (sphingoid) backbone

• 15-tetracosenoic acid• Fatty acyl group

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Mol. BioSyst., 2015, 11, 698--713 | 699

Structures and Nomenclature

Ceramide Biosynthesis

Front. Endocrinol., 30 July 2014 | https://doi.org/10.3389/fendo.2014.00127

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Ceramide Biochemistry

Ceramides are found in high concentration in the membrane of cells

• Structural component of the lipid bilayer

• Bioactive lipid - implicated in a variety of physiological functions including:

• Apoptosis and cell growth arrest

• differentiation and cell senescence

• cell migration and adhesion

Ceramides are converted rapidly to more complex sphingolipids:

• Sphingomyelin

• Glycosylceramides

• Little accumulation observed

– Except for the skin (50% of total lipids can be ceramides)

Biosynthesis of Ceramides

De novo biosynthesis

• Ceramide synthases couple sphinganine + long chain fatty acid to form

dihydroceramide

• Double bond introduced into position 4 of the sphingoid base

ceramide synthases 5 and 6 generate are specific for palmitic acid

ceramide synthases 1 (brain and skeletal muscle) specific for stearic acid

ceramide synthases 2 specific for very long chain CoA-thioesters (C20-C26)

ceramide synthases 3 unusual ceramides of skin & testes

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Biosynthesis of Ceramides

Catabolism of complex sphingolipids:

• Sphingomyelinases/phospholipase C breakdown sphingomyelin in

animal tissues

• Many factors can stimulate the hydrolysis of sphingomyelin to

produce ceramide:

Cytokines :TNF-a, IFN-g & various interleukins

1,25-dihydroxy-vitamin D3

endotoxin

nerve growth factor

ionizing radiation & heat

LC Method DevelopmentWhere to Start?

• Designing and optimizing an LC method involves choosing appropriate:

1. Separation mechanism: NPC, RPLC, HILIC, size exclusion ion, exchange etc

2. Column chemistry: C2, C4, C8, C18, cyanopropyl, phenyl, biphenyl, amide, SiOH etc

3. Column properties: pore size, particle size & column dimensions

4. Stationary and mobile phase combinations

• Critical to optimizing the chromatographic efficiency, retention, resolution & selectivity of analytes

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Ceramide Scouting Gradients on Waters BEH C18

Steep gradient; large change in organic modifier.

Provides initial information about analyte retention & resolution.

Aids in expediting the development of an optimized LC method

Poor Resolution (R)

Long Retention (Tr) Time

1. C16:02. C18:13. C18:04. C20:05. C24:16. C22:07. C24:0

1 2 3 4 7

5 6

Fractionation of Ceramide Metabolites on Waters CSH C18 Column

Column: 100x2.1mm 1.7u Solvent System:A= MeCN:water (60:40 v/v)B= 2-propanol:MeCN (90:10 v/v)10mM NH4OAc + 0.1% formic acidBallistic gradient0.4ml/min @ 55oC

UPLC-ESI-QTOF-MS

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0 5 1 0 1 5 2 0

2 0

4 0

6 0

8 0

1 0 0

G ra d ie n t C o m p a r is o n

t im e (m in )

% o

rga

nic

b a llis t ic

s ta n d a r d

Ballistic Gradient:

• Ultra-fast gradient

• Rapid change in organic modifier

• Separations performed at high flow rates

• High linear velocity

Fractionation of Ceramide Metabolites on Waters CSH C18 Column

1. C16:02. C18:13. C18:04. C20:05. C24:16. C22:07. C24:0

UPLC-ESI-MS-MS

Column: 100x2.1mm 1.7u Solvent System:A= MeCN:water (60:40 v/v)B= 2-propanol:MeCN (90:10 v/v)10mM NH4OAc + 0.1% formic acidBallistic gradient0.4ml/min @ 55oC

QQQ MS Detector

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Ceramide Extraction

Extraction protocols and LC-MS methodsadapted from Shaner RL et al JLR 2009

• Add 50 mg of liver tissue to 50% aqueous methanol– Why not chloroform directly?

• Homogenize in Bertin Precellys at 6500 rpm with ~10 zirconium beads for 30 seconds

Ceramide Extraction – cont’d

• Add 1 mL of CHCl3:MeOH (2:1, v/v) containing 20 μl of C17:0 internal standard solution (use 1 mM stock solution)– Why internal standard at this point?– Should we use glass or plastic? Does it matter?– What if you swap chloroform for hexane or isopropanol?

• Homogenize again and centrifuge at 18,000xg for 10 min to separate phases

• Transfer organic phase to a new tube (#2) and repeat extraction of left over material – Why repeat?

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• Combine organic phases and dry down in a vacuum centrifuge

• Solubilize residuals in 50 μl of CHCl3:MeOH (2:1, v/v)– Why chloroform here?

• Saponification or acid hydrolysis of residuals to release ceramides

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• Incubate residuals with 0.5 mL of 1M HCl in MeOH @ 50°C for 1 hr (or base for sapn)

• Cool samples and re-extract

• Solubilize with in 30 μl of CHCl3:MeOH (2:1, v/v), sonicate for 5 minutes in sonicatingwater bath– Why sonicate?


• Dilute 10 fold with acetonitrile:isopropanol:water (1:1:1, v/v)

• Centrifuge to remove any particulates and transfer to autosampler tube

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0

11 0 3

21 0 3

31 0 3

41 0 3

E ffe c t o f S o lv e n t S y s te m o n C 1 6 :0 C e ra m id e R e c o v e ry fro m M u r in e L iv e r

C 1 6 :0 C e ra m id e

pe

ak

are

a/m

g l

ive

r t

iss

ue

C H C l3 :M e O H

E tO A c

H e x a n e :E tO A c

H e x a n e :IP A

M tB E

0

11 0 3

21 0 3

31 0 3

41 0 3

E ffe c t o f S o lv e n t S y s te m o n C 2 4 :0 C e ra m id e R e c o v e ry fro m M u r in e L iv e r

C 2 4 :0 C e ra m id e

pe

ak

are

a/m

g l

ive

r t

iss

ue

C H C l3 :M e O H

E tO A c

H e x a n e :E tO A c

H e x a n e :IP A

M tB E

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Ileum Serum

Lipidomics Reveals Ceramides are Decreased inFXR Intestine-null Mice

J Lipid Res. 2011 Aug; 52(8): 1583–1594. doi: 10.1194/jlr.D015586

Ceramide Labeling

0500

10001500200025003000

1 2 3 4

C16 cer

C16 Cer C16 Cer+16 C16 Cer+32

0

5000

10000

15000

20000

25000

1 2 3 4

c16sm

c16sm c16 sm+16 c16 sm+32

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Direct energy source

Anabolic substrates

Regulatory roles

Short Chain Fatty Acids• SCFAs are those carboxylic acids that contain aliphatic tails less than 6 carbon atoms

• In humans, SCFAs are derived in large part from fermentation of carbohydrates and proteins in the colon

• By this process, the host is able to salvage energy from foods that cannot be processed normally in the upper parts of the gastrointestinal tract

• SCFAs serve as direct energy source, anabolic substrates and signaling molecules involve metabolic and immunological regulation.

SCFA Extraction and Detection

• What do we know about SCFAs?

• How do we detect them?

• What problems might influence SFCA measurement?

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3 .0 0 4 .0 0 5 .0 0 6 .0 0 7 .0 0 8 .0 0 9 .0 0 1 0 .0 0 1 1 .0 0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

3 5 0 0 0 0

4 0 0 0 0 0

4 5 0 0 0 0

5 0 0 0 0 0

5 5 0 0 0 0

6 0 0 0 0 0

T im e -->

A b u n d a n c e

T IC: JC1 6 0 1 1 5 _ 0 6 .D \ d a ta .m s

aceticacid

butyricacid

propionicacid

pyridine

aceticacidbutyricacidpropionicacid

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

T ime-->

Abundanc e

T IC: JC160606_new 13.D \ data.ms

caproic acid-6,6,6-d3(IS)

caproic acid-6,6,6-d3(IS)

carbonicacid

Total ion chromatogram of 100 µg/ml

SCFAs standard mixture with 10

µg/ml caproic acid-6,6,6-d3 as

internal standard (IS) by (top) propyl

esterification method and (bottom)

acidified water method.

Notice change in retention time

between the two methods.

Derivatized

Native

2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 1 0 5 1 1 00

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

1 2 0 0 0 0

1 4 0 0 0 0

1 6 0 0 0 0

1 8 0 0 0 0

2 0 0 0 0 0

2 2 0 0 0 0

2 4 0 0 0 0

2 6 0 0 0 0

m / z -->

A b u n d a n c e

A v e ra g e o f 2 .7 2 2 to 2 .7 3 8 m in .: JC 1 6 0 1 1 5 _ 2 9 .D \ d a ta .m s4 4 .0

6 2 .0

7 4 .0

3 1 .0

8 7 .95 4 .9 1 0 1 .9

2.60 2.65 2.70 2.75 2.80 2.85 2.90 2.950

50000

100000

150000

200000

250000

300000

Time-->

Abundance

Ion 44.00 (43.70 to 44.70): JC160115_29.D\ data.ms

2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 1 0 5 1 1 00

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

1 2 0 0 0 0

1 4 0 0 0 0

1 6 0 0 0 0

1 8 0 0 0 0

2 0 0 0 0 0

2 2 0 0 0 0

2 4 0 0 0 0

2 6 0 0 0 0

2 8 0 0 0 0

3 0 0 0 0 0

3 2 0 0 0 0

3 4 0 0 0 0

3 6 0 0 0 0

m / z - - >

A b u n d a n c e

A v e r a g e o f 2 . 7 2 8 t o 2 . 7 4 1 m in . : J C 1 6 0 1 1 5 _ 0 7 . D \ d a t a . m s4 3 . 0

6 1 . 0

7 3 . 0

3 1 . 0

8 7 . 05 3 . 0 1 0 0 . 82.5 5 2 .60 2 .65 2 .7 0 2 .75 2 .80 2 .85 2 .90 2 .950

5000 0

1 0000 0

1 5000 0

2 0000 0

2 5000 0

3 0000 0

3 5000 0

4 0000 0

T ime -->

A bu ndanc e

Ion 4 3 .00 (42 .70 to 4 3 .70 ): JC160115_ 07 .D \ da ta .ms

propylacetate(aceticacid)

1-13Cpropylacetate(1-13Caceticacid)

13C

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SCFA by 1H NMR

ppm

0.70 .80 .91 .01 .11.21 .31 .41 .51 .61 .71 .81 .92 .02 .12 .2 ppm

15

20

25

30

35

40

acetaten-butyrate

n-butyrate

n-butyrate

propionatepropionate

Acetate#1

Propionate#1

Propionate#2

Butyrate#1

Butyrate#2

Butyrate#3

1H-13C HSQC NMR

spectroscopy of mice feces.

Reference spetra were

obtained from Human

Metabolome Database

(HMDB). Acetate

(HMDB00042), Propionate

(HMDB00237) and Butyrate

(HMDB00039)

GC-MS Propyl Esterification Method

CompoundsSpiked amount

(µg/ml)

Amount recovered

(µg/ml)%Recovery %RSD %Matrix effect

Acetic Acid

10 6.7±1.0 66.9 14.9 76.3

100 108.3±15.0 108.3 13.9 94.4

250 254.5±16.3 101.8 5.9 92.7

Propionic Acid

10 7.2±1.2 72.3 16.9 74.2

100 100.2±17.6 100.2 17.5 84.2

250 244.5±36.6 97.8 15.0 92.7

Butytic Acid

10 6.7±1.1 67.0 11.2 77.7

100 99.4±20.4 99.4 20.6 88.2

250 249.7±38.1 99.8 15.3 97.4

GC-MS Acidified Water Method


(µg/ml)

Amount recovered


Acetic Acid

10 7.5±1.9 74.8 25.1 116.8

100 108.7±14.9 108.7 13.7 95.7

250 226.1±19.2 90.4 8.5 76.2

Propionic Acid

10 7.5±1.2 75.4 16.0 95.1

100 106.3±10.7 106.3 10.1 80.3

250 297.2±23.1 118.9 7.8 76.2

Butyric Acid

10 5.2±0.4 52.2 6.8 89.6

100 87.6±17.0 87.6 20.6 76.2

250 291.7±24.3 116.7 8.3 79.7

1H NMR Quantification Relative to TSP Method


(µg/ml)

Amount recovered


Acetic Acid

10 8.4±0.4 83.7 4.2 90.6

100 97.7±4.3 97.7 4.4 118.5

250 271.7±16.0 108.7 5.9 111.7

Propionic Acid

10 10.1±0.6 101.4 6.0 107.2

100 116.6±5.5 116.6 4.7 116.1

250 332.1±15.3 132.8 4.6 104.8

Butyric Acid

10 5.4±0.8 54.2 14.7 64.1

100 92.9±4.6 92.9 5.0 118.1

250 263.2±14.7 105.3 5.6 106.8

1H NMR Quantification with Calibration Curve Method


(µg/ml)

Amount recovered

(µg/ml)%Recovery %RSD

Acetic Acid

10 8.6±0.4 85.6 4.2

100 98.7±5.1 98.7 5.2

250 277.9±16.3 111.2 5.9

Propionic Acid

10 8.0±0.48 80.2 6.0

100 91.4±4.6 91.4 5.0

250 262.7±12.1 105.1 4.6

Butyric Acid

10 5.4±0.8 54.1 14.7

100 92.0±4.7 92.0 5.1

250 263.0±14.7 105.2 5.6

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GC-MSPropylEsterificationMethod

Compounds Intraday(%RSD) Interday (%RSD)

10µg/ml 25µg/ml 100µg/ml 250µg/ml 10µg/ml 25µg/ml 100µg/ml 250µg/ml

AceticAcid 4.3 2.7 6.1 3.0 4.5 3.5 10.0 3.4

PropionicAcid 12.2 5.7 7.7 5.6 21.3 6.5 11.2 8.4

ButyricAcid 1.2 3.5 5.8 3.6 7.7 5.1 4.6 6.9

GC-MSAcidifiedWaterMethod



AceticAcid 12.6 6.8 5.1 4.5 9.8 5.6 5.7 5.3

PropionicAcid 6.4 7.2 4.9 5.4 8.9 7.9 7.8 7.9

ButyricAcid 2.4 4.2 5.0 5.5 4.7 6.4 6.0 7.7

1HNMRQuantificationRelativetoTSP



AceticAcid 1.7 4.6 0.3 1.9 2.6 5.3 0.3 0.6

PropionicAcid 3.8 0.9 2.4 2.8 1.4 2.4 1.4 2.0

ButyricAcid 4.3 4.5 1.4 2.1 4.5 2.6 0.7 4.0

1HNMRQuantificationwithCalibrationCurve



AceticAcid 1.1 6.7 1.7 1.8 1.6 4.3 0.3 0.6

PropionicAcid 2.0 0.7 2.3 2.7 0.7 1.7 1.3 1.9

ButyricAcid 2.3 3.2 1.3 2.1 2.4 1.9 0.7 3.9

How do the methods compare?

0

400

800

2000

4000

µg

/gF

ec

es

GC-MS Propyl Esterification Method

GC-MS Acidified Water Method1H NMR Quantification Method

Fecal SCFAs Concentration

NS

NS

NS

Biological concentrations of SCFAs in mice fecal samples measured by GC-MS propyl

esterification method, GC-MS acidified water method and 1H NMR quantification

method. Values are expressed as the mean ± 95% CI. (n=10). One-way ANOVA.

Biological Concentrations measured by different methods were not significantly

different.

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SCFA Measurement in the Real World

0

2

4

50

100

200

1400

2600

3800

µg

/gGF

CONV-R****p<0.0001

****p<0.0001 ****p<0.001

Biological concentrations of

SCFAs in germ free(GF) and

conventionally raised (CONV-R)

fecal samples measured by GC-

MS propyl esterification

method. Values are expressed

as the mean ± sd. (n=5). Two-

tailed t-test.

Conclusions

• Extraction protocols can impact metabolomic data sets considerably

• Solvent system composition and pH exhibit the most dramatic effects on metabolite recovery – The magnitude of these effects depend on metabolite

class

– Some classes of metabolites

• The number of extraction repetitions also plays a role in enhancing metabolite recovery– Tradeoff - longer sample prep time

– Larger sample volumes to process (evaporate)

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Conclusions

• Traditional RPLC methods can provide efficient separation of acyl-carnitine, bile acid and CoA thioester mixtures.

– Advancements in hybrid particle technologies

– Allowing for extremes in mobile phase pH and temperature – manipulate selectivity

– Complex ligand stationary phase interactions

• HILIC methods are superior at separating highly polar metabolites.

– Nucleotides and derivatives

– Small polar metabolites – sugars, organic acids, amino acids, hydrophilic vitamins

Conclusions – cont’d

• There’s no one “perfect” extraction or LC method available capable of efficiently extracting or resolving, respectively, all components or features in the metabolome

• Advanced column chemistries (amide, aminopropyl, biphenyl, graphite, phenyl-hexyl) and alternative chromatographic methodologies (HILIC) can provide enhanced coverage of the metabolome

• Different platforms can provide greater confidence in metabolite measurement

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Acknowledgments

Penn State University

• Chris Chiaro

• Philip Smith

• Jared Correll

• Jingwei Cai

• Jingtao Zhang

National Cancer Institute

• Frank J. Gonzalez

• Kris Krausz

• Changtao Jiang

• Fei Li

NIEHS R01 ES022186

MRC

• Julian Griffin

• Elizabeth Stanley

Improving the Metabolomics Mass Spectrometry Workflow ... · •Most systems use either liquid or...

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