Metabolomics & Lipidomics: From Discovery to Routine Applications

©2015 Waters Corporation 1

Metabolomics and Lipidomics:

from Discovery to Routine Applications

11th International Conference of the Metabolomics Society

June 29th 2015 to July 2nd 2015

Giuseppe Astarita Principal Scientist, Health Sciences

Waters Corp, USA


Metabolomics and Lipidomics Application Notebook – Waters Corporation, April 2015. Literature part number 720005245EN – http://www.waters.com/waters/library.htm?cid=511436&lid=134841740

Compilation of Recent Work

http://www.waters.com/waters/library.htm?cid=511436&lid=134841740

In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology)

Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards)

Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure)

O

O

HO

R1 O ON

+P

OH

O

O

R2

Phosphotadylcholine (PC)

sn-1

sn-2

sn-1-H20

sn-2-H20

Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating)

1 2 3 4

Metabolomics Approaches




O

O

HO

R1 O ON

+P

OH

O

O

R2


sn-1

sn-2

sn-1-H20

sn-2-H20


1 2 3 4


Measure (Mass Spectrometry)

Separate (Chromatography; Ion Mobility)

Process and Mine (Informatics and Statistics)

Untargeted Metabolomics and Lipidomics

Metabolomics: UPLC Separation

Paglia G.et al. Anal Chem 2014

Low Energy

High Energy

Metabolomics: APGC Separation

Arabidopsis

Courtesy of Prof Vladimir Shulaev

Lipidomics: UPLC Separation ChoE & TG PC, SM, PG, PE

lysophospolipids SM, DG, ChoE

PC, PG,PI, PS, PE

Free Fatty Acids

Damen C., et al. Journal Lipid Research 2014

Lipidomics: HILIC/SFC

Mouse heart extract

Miroslav Lísa and Michal Holcapek Anal Chem. 2015 Jun 20




O

O

HO

R1 O ON

+P

OH

O

O

R2


sn-1

sn-2

sn-1-H20

sn-2-H20


1 2 3 4


Drift time

m/z

Ceramide species

Drift time (ms)

Drift time (ms)

p _ _ _ _ _

Drift

tim

e (m

s)

Retention time (min)

Rel

ativ

e In

tens

ity

(%)

0 2 4 6 8 10 12 14 16 18 20

ChoE & TG PC, SM, PG, PE lysophospholipids

Orthogonal Coordinates

Paglia G., et al. Anal Bioanal Chem 2015

Paglia G.et al. Anal Chem 2015; Paglia et al. Anal Chem 2014

Ion Mobility Separation: Hybrid Q-TOF

Poster 353 – The Analysis of Bile Acids: Enhancement of Specificity Using Travelling Wave IMS-QTof Mass Spectrometry





DIA with IMS (HDMSE)

Paglia G., et al. Anal Bioanal Chem 2015

Composite ion map after peak picking

Individual ion maps

Data alignment and peak picking

ANOVA filtering and Multivariate Statistics

Database search

Filtering by ANOVA P value

Control Radiation

Control

Radiation

Tentative Identifications

1.

2. 3.

4.

5.

Data Processing and Mining with Progenesis QI Software

Laiakis E. et al, J Proteome Res. 2014 Sep

http://www.waters.com/waters/nav.htm?cid=134790655

0

20

40

60

80

100

120

140

160

180

5 6 7 8 9 10 11 12 13 14 15

iden

tific

atio

ns

ppm m/z error

HMDB In house database no RT In house database with RT <0.6 min

Database Search Using Orthogonal Coordinates

Database Search Using Orthogonal Coordinates

HDMSE

<25 fragments HDMSE High Energy

HDMSE Low Energy 750.5422

750.5422 303.2324 (FA 20:4)

OHO

O ONH2

PHO

O

O

303.2324 (FA 20:4)

%

%

m/z 200 1400

With Ion mobility separation

Co-eluting ions

Fragments Transferred to TOF-MS

Fragmentation

Methodological References: Untargeted

A Facile Database Search Engine for Metabolite Identification and Biomarker Discovery in Metabolomics Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC/QTof MS Lipid Separation Using UPLC with Charged Surface Hybrid Technology (pending publication)












O

O

HO

R1 O ON

+P

OH

O

O

R2


sn-1

sn-2

sn-1-H20

sn-2-H20


1 2 3 4


Analyze (Software)

Measure (Tandem Mass Spectrometry)

Separate (Chromatography)

Sample Preparation (extraction Liq/Liq; SPE)

Targeted Metabolomics

Targeted Metabolic Profiling: Metabolomics kits

Metabolite group No. of metabolites

Amino acids and Biogenic amines

40

Acylcarnitines 40

Lyso-phosphatidylcholines 14

Phosphatidylcholines 74

Sphingomyelins 14

Hexose 1

Total 183

+

Laiakis E. et al, J Proteome Res. 2014 Sep

Biocrates AbsoluteIDQ p180 Kit

Waters Xevo TQ-S Mass Spectrometer

http://www.biocrates.com/products/research-products/absoluteidq-p180-kit

http://www.biocrates.com/products/research-products/absoluteidq-p180-kit



Eicosanoids: Bioactive Oxygenated PUFAs

Omega-6 metabolites: pro-inflammatory

Omega-3 metabolites: anti-inflammatory

Astarita et al. Biochim Biophys Acta. 2015 Apr;1851(4):456-468

Plasma sample

Add internal standard mix

Load

Inject into UPLC/MS-MS

SPE clean up

Multiplexed Assay for Eicosanoids Profiling

Selected Lipid

Internal Standard

Methodological References: Targeted

Targeted Lipidomics of Oxylipins (Oxygenated Fatty Acids)

Targeted Lipidomics Using the ionKey/MS System

Targeted Metabolomics Using the UPLC/MS-based AbsoluteIDQ p180 Kit














O

O

HO

R1 O ON

+P

OH

O

O

R2


sn-1

sn-2

sn-1-H20

sn-2-H20


1 2 3 4




100 700 1500 m/z

50

150

Dri

ft T

ime

(bin

s)

+

+

+

Ion mobility MS

Brain slice

Excitation source (i.e., laser, ESI)

+

Scanning tissues

along the axes

desorption/ionization

MOLECULAR HISTOLOGY

Paglia G.et al. Anal Chem 2015

MS Imaging with Ion Mobility


In Situ Metabolomics

REIMS Research System with iKnife for Direct Sampling


Methodological References: MS Imaging

Improved MALDI Imaging Quality and Speed Using the MALDI SYNAPT G2-Si HDMS

Biomarker Discovery Directly from Tissue Xenograph Using High Definition Imaging MALDI Combined with Multivariate Analysis

Data Independent MALDI Imaging HDMSE for Visualization and Identification of Lipids Directly from a Single Tissue Section

Utility of Desorption Electrospray Ionization (DESI) for Mass Spectrometry Imaging


















Peer-Reviewed References

Conclusions

Spatial Localization/Real Time

Quant/Qual applications

Screening and Identify

O

O

HO

R1 O ON

+P

OH

O

O

R2


sn-1

sn-2

sn-1-H20

sn-2-H20

Chemical Diversity

1 2 3 4


Acknowledgements

Jon Williams Scott Geromanos

Donald Mason Steven Lai

Giorgis Isaac Hernando Olivos

Emmanuelle Claude Jim Langridge

Arthur Moseley Will Thompson

David Grant Lochana Menikarachchi

Giuseppe Paglia Bernhard O. Palsson

Skarphédinn Halldórsson Ottar Rolfsson

Peggi Angel Callee Walsh Greg Kilby

Thomas Hankemeier Rob Vreeker

Katrin Strassburg

Albert Joseph Fornace Evagelia C. Laiakis

Ralf Bogumil Cornelia Roehring

Therese Koal

Jing Kang Jennifer McKenzie