EMBO Practical Course on Metabolomics Bioinformatics for Life Scientists Oscar Yanes, PhD “Dissecting an untargeted metabolomic workflow”

EMBO Practical Course on Metabolomics Bioinformatics for Life Scientists

Oscar Yanes, PhD

“Dissecting an untargeted metabolomic workflow”

Untargeted metabolomics workflowUntargeted metabolomics workflow

HypothesisExperimental validation

Samplepreparation

Sample analysisby MS and NMR

Pre-processingdata analysis

Metaboliteidentification

Experimentaldesign



Samplepreparation




EMBO Course

Experimentaldesign

Hypothesis

Biomarker discovery

List of metabolites differentiallyregulated

Pathway analysis Model construction Scientific literatureDisease vs. control

Validation

Mechanism

Ultimate goal of metabolomics



Samplepreparation




Experimentaldesign

THE IMPORTANCE OF EXPERIMENTAL DESIGN

COLLABORATOR

I want to do metabolomicsI want to do

metabolomics

ME


COLLABORATOR

I want to do metabolomicsI want to do metabolomics

ME

……

I have many samples at -80°C.

Could you do metabolomics and

find out something?



find out something?


COLLABORATORME



find out something?



find out something?

!!!!


COLLABORATORME


BASIC DIAGRAM OF A MASS SPECTROMETER


Gas-phase:Gas chromatography

Liquid-phase:Liquid chromatographyCapillary electrophoresis

Solid-phase:Surface-based


Electron ionization (EI)Chemical ionization (CI)Atmospheric pressure chemical ionization (APCI)Electrospray ionization (ESI)Laser desorption ionization (LDI)

Glucose

0.0

0.1

0.2

0.3

0.4

0 4 12 24Time (h)

Are

a/A

rea

(IS

)

Lactate

0.0

0.2

0.4

0.6

0.8

1.0

0 4 12 24Time (h)

Are

a/A

rea

(IS

)

Pyruvic Acid

0.0

0.1

0.2

0 4 12 24Time (h)

Are

a/A

rea

(IS

)

Choline

0.0

0.2

0.4

0.6

0.8

1.0

0 4 12 24Time (h)

Are

a/A

rea

(IS

)

Watch out serum/plasma samples from biobanks!Watch out serum/plasma samples from biobanks!



Samplepreparation

Sample analysisby MS



Experimentaldesign

Maximize ionization efficiency over the whole mass range (e.g., m/z 80-1500)

Requisite for untargeted metabolomics


Number of features Intensity of the features



Number of features Intensity of the features

Coverage of the metabolome Accurate quantification and identification of metabolites


How do we increase the number of features and their intensity??

time

massintensity

Feature: molecular entity with a unique m/z and retention time value

How do we increase the number of features and their intensity??

time

massintensity

Sample preparation: - Extraction method

Chromatography: - Stationary-phase- Mobile-phase

Ion Funnel Technologyetc.

Hot EtOH/Amm. Acetate Cold Acetone/MeOH

Only 45% of the metabolites are detected with Acetone/MeOH

MS/MS threshold

Extraction method

Yanes O., et al. Anal. Chem. 2011; 83(6):2152-61

Extraction method

Liquid Chromatography: mobile-phase

Yanes O et al. Anal. Chem. 2011; 83(6):2152-61

Ammonium Fluoride Ammonium acetate Formic acid

Ammonium fluoride

Ammonium acetate

F-

Ammonium fluoride

Chromatography: stationary phase

HILIC RP C18/C8

LC flow rate and pressure: UPLC vs. HPLC vs. nanoLC (vs. GC!)

HPLC

UPLC

MinutesMinutes

Effect of pH; ammonium salts; ion pairs (e.g. TBA)


Electron ionization (EI)Chemical ionization (CI)Atmospheric pressure chemical ionization (APCI)Electrospray ionization (ESI)Laser desorption ionization (LDI)

PRACTICAL ASPECTSPRACTICAL ASPECTS

1. Number of scans/secondImplications in LC/MS and GC/MS:

QuantificationMaximum intensity or integrated area

2. Instrument resolutionImplications:

Detector saturationQuantification

3. Sample amount injectedImplications:

Detector saturation



Samplepreparation




EMBO Course

Experimentaldesign

RAW METABOLOMICS DATA

PRE-PROCESSINGPRE-PROCESSING

STATISTICAL ANALYSISSTATISTICAL ANALYSIS

RAW DATA CONVERSIONRAW DATA CONVERSION

METABOLITE IDENTIFICATIONS


FROM RAW DATA TO METABOLITE IDs

PRE-PROCESSING

PRE-PROCESSING

STATISTICAL ANALYSIS


RAW DATA CONVERSION

RAW DATA CONVERSION



PATHWAY ANALYSISPATHWAY ANALYSIS

FROM RAW DATA TO METABOLITES IDs

LC/MSGC/MS

GC/MSLC/MS

LC-MS RAW DATA

LC-MS RAW DATA

LC-MS WORKFLOW

PREPROCESSING


mZDATA

IDENTIFICATION

............

...I...M2

......IM1

mZRT3mZRT2mZRT1

mZRT2M2

mZRT1M1

mZRT Features Table

Feature: individual ions with a unique mass-to-charge ratio and a unique retention time

PROTEOWIZARD

LC-MS WORKFLOW

RAW LC-MS DATA TO mZXML: PROTEOWIZARD

[Nature Biotechnology, 30 (918–920) (2012)]

VENDOR FORMATS CONVERTERAgilent MassHunter.d ProteoWizardBruker Compass.d, YEP, BAF, FID ProteoWizardThermo Fisher RAW ProteoWizardWaters MassLynx.raw ProteoWizardAB Sciex WIFF ProteoWizard

LC-MS WORK-FLOW

XCMS PRE-PROCESSING

•http://metlin.scripps.edu/download/•Free & Open Source•Based on R•On-line version

•Suitable for:-GC-MS-LC-MS

Analytical Chemistry, 78(3), 779–787, 2006Analytical Chemistry, 84(11), 5035-5039, 2012

http://metlin.scripps.edu/download/

LC-MS WORKFLOW

[BMC Bioinformatics, 2008 9:504]

XCMS PRE-PROCESSING

1. FEATURE DETECTION

LC-MS WORKFLOW

XCMS PRE-PROCESSING

1. FEATURE DETECTION

1. Dense regions in m/z space2. Gaussian peak shape in chromatogram

LC-MS WORK-FLOW

XCMS PRE-PROCESSING

2. RETENTION TIME CORRECTION

LC-MS WORKFLOW

FEATURES RANKINGThose features varying according to our phenomena are retained to further identification experiments


• 103-104 mZRT features IDENTIFICATION NOT FEASIBLE!• features redundancy:

-adducts: [M+H+], [M+Na+], [M+NH4+], [M+H+-H2O]…

-isotopes: [M+1], [M+2], [M+3]• Many mZRT features are noisy in nature and irrelevant to our phenomea

LC-MS WORK-FLOWFEATURES RANKING CRITERIA

WORKLIST

-RANDOMIZE-USE QCs TO CHECK ANALYTICAL VARIATION

(I) ANALYTICAL VARIABILITY

LC-MS WORK-FLOW

FEATURES RANKING CRITERIA

(I) ANALYTICAL VARIABILITY

100)(

)(

)(

QCmZRT

QCmZRTQC

mZRT

j

j

j X

SCV

100)(

)(

)(

TmZRT

TmZRTT

mZRT

j

j

j X

SCV

USEFUL PLOTS IN EXPLORATORY DATA ANALYSIS

NEURONAL CELL CULTURESKO (N=15) vs WT (N=11)#mZRT=6831

RETINASHypoxia (N=12) vs Normoxia (N=13)#mZRT=7654

LC-MS WORK-FLOWFEATURES RANKING CRITERIA

(IV) HYPOTHESIS TESTING+FDR

=0.05 (235 features significantly varied by chance, 26% out of 900)

FDR=0.0074 (20 features varied by chance, 5% out of 404)

#features=4704







LC-MS WORKFLOW

(i) analytical variability

(ii) features intensity

# mZRT=51908

# mZRT=38377

# mZRT=4704

# mZRT=250

(iii) hypothesis testing + fold change

10M data points

Annotation

Data Base look-up

Identification experiments

10-50 differential metabolites

Workflow for Metabolite Identification

Step 1: Select interesting featuresStep 1: Select interesting features

Step 2: Search databases for accurate massStep 2: Search databases for accurate mass

Step 3: Filter “putative” identification listStep 3: Filter “putative” identification list

Step 4: Compare RT and MS/MS of standardsStep 4: Compare RT and MS/MS of standards

Step 1: Select interesting featuresStep 1: Select interesting features

Step 2: Search databases for accurate mass

Step 3: Filter “putative” identification list

Step 4: Compare RT and MS/MS of standards


Step 1: Select interesting features






HMDB MetlinEach feature returns many hits.


Common adducts Na+, NH4+, K+, Cl-, and H2O loss

Adducts increase number of hits returned!







Eliminate•drugs?• intensity in the mass spectrum• adducts?• matches with obviously inconsistent retention times

Example: feature with m/z 733.56 is unlikely to be a phospholipid if it has a 1-min RT with reverse-phase chromatography.

Look for hits that implicate the same pathway, give those features priority.Look for hits that implicate the same pathway, give those features priority.

Standards can be expensive, your intuition will save you money and time!







What experimental data should be required to constitute a metabolite identification?

• Accurate mass?

• Retention time?

• MS/MS data?

Unlike proteomics, no journals have requirements or guidelines for publication of metabolite identifications.

“The identification of certain metabolites as their exact masses in their given biological context was strategic in the context of searching for biomarkers for CD.”

accurate mass

“…this method enables untargeted profiling of metabolites using accurate mass-retention time (AMRT) identifiers.”

accurate mass and retention time

“Metabolites were putatively identified on the basis of accurate mass and retention time, and confirmed by comparing MS/MS data of unknowns to model compounds.”

accurate mass, retention time, and MS/MS


accurate mass

Accurate mass identifications are putativeAll structures have a neutral mass of 146.0691

Mass error (even if small) and adducts add more possibilities!


accurate mass

“…this method enables untargeted profiling of metabolites using accurate mass-retention time (AMRT) identfiers.”






Many structural isomers have the retention time

Citrate and isocitrate have the same retention time but different MS/MS patterns.

isocitrate

citrate


accurate mass







Mass-to-Charge (m/z)60 100 140 180 220 260 300 340 380 420

367.33

367.33

H

H

H

HO

H

H

OH

Standard7α-hydroxy-cholesterol

Biological sample

Q-TOF


Retention time will be available from the profiling experiment, however, to obtain MS/MS data for the feature of interest in the research sample typically another experiment is required.

Note: Only need to perform MS/MS on one research sample. Pick a sample from the group for which the feature is up-regulated!

Do not pick this group


What if feature of interest is not in the database?(or model compound is not commercially available)

FT-ICR MS can be used to limit chemical formulas

MS/MS can be insightful to reveal structural insight(MS/MS library, bioinformatic approaches)

NMR can provide structural details

When a chemist is your best friend…
















• Thermophile organism adapted to live at high temperatures.

• Organisms challenged with cold temperature (72 º C) and compared to high-temperature (95 º C) controls.

Feature up-regulated at cold temperature

Identification???

Natural productNatural product

N1-AcetylthermospermineN1-Acetylthermospermine

*

*

Feature up-regulated at cold temperature



*

*

Intensity of m/z 112 fragment is significantly different. NOT A MATCH!

Chemical synthesis of hypothesized structure is required

Synthesized metabolite produces comparable MS/MS data as natural product from Pyrococcusfuriosus.



N4(N-Acetylaminopropyl)spermidineN4(N-Acetylaminopropyl)spermidine

Hypothesis

Biomarker discovery

List of metabolites differentiallyregulated

Pathway analysis Model construction Scientific literatureDisease vs. control

Validation

Mechanism

Ultimate goal of metabolomics

LC and GC-Triple quadrupole MS

Validate your metabolites!!

Targeted metabolomics Molecular biology techniques

ImmunohistochemistryReverse Transcription-PCRGene expression arrayCell culturesAnimal experimentation …..

Thank you

email: [email protected]: www.yaneslab.comTwitter: @yaneslab

http://www.yaneslab.com/

Documents

EMBO Practical Course on Metabolomics Bioinformatics for Life Scientists Oscar Yanes, PhD “Dissecting an untargeted metabolomic workflow”