Universitätsklinikum Regensburg

Guidelines for Lipidomics Analysis and Reporting

– the Lipidomics Standards Initiative (LSI)

7th June 2018

Gerhard Liebisch1, John A. Bowden2, William J. Griffiths3, Robert Ahrends4, Todd W. Mitchell5,

Makoto Arita6, Christer S. Ejsing7, Michal Holčapek8, Markus Wenk9 and Kim Ekroos10

1 Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany. 2Chemical Sciences Division, Hollings Marine Laboratory, National Institute of Standards and Technology, USA

3 Swansea University Medical School, ILS1 Building, Singleton Park, Swansea, UK. 4 Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany

5 School of Medicine, Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia 6 Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences (IMS), Tsurumi, Yokohama, Kanagawa 230-0045, Japan

7 Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark. 8 Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.

9 Singapore Lipidomics Incubator (SLING), Department of Biochemistry, YLL School of Medicine, National University of Singapore, Singapore, Singapore. 10 Lipidomics Consulting Ltd., FI-02230 Esbo, Finland.

MISIDENTIFICATION

Why is there a need for standardization?

Metabolomics discovers novel “Lipid biomarker”

Samples

n = 600

Methanol

precipitation

UPLC (RP) -HighRes

MS pos/neg ion mode

IS

QC samples (n=28 per 100)

Signal correction

using QC samples

Annotation by

PUTMEDID-LCMS

Inverse association of PC O-20:0/O-20:0 and

LPC dm16:0 with T2D

Drogan et al. Clin

Chem. 2015

Mar;61(3):487-97

Information provided by Drogan et al. (1)

LIPID MAPS

database search1

Reported

m/z RT [s] Reported lipid species Adduct ion

Appropriate lipid

species annotation2 Expected m/z values3

Negative ion mode [M-H]- [M+HCOO]- [M+35Cl]-

no match 860.684 988 PC(O-20:0/O-20:0) ? PC dO-40:0 862.727 852.698

PC O-34:3

[M+HCOO]-

786.5654

786.564 914

GlcCer(d18:0/18:0) ? HexCer d36:0 728.605 774.610 764.581

PC(16:0/O-16:0)

PC(O-14:0/18:0)

PC(O-16:0/16:0)

? PC O-32:0 764.581 754.552

PC(O-16:0/18:3)

PC(O-16:0/18:3)

PC(P-16:0/18:2)

? PC O-34:3 786.565 776.537

PC(16:1/dm18:1)

PC(16:1/dm18:1)

PC(18:2/dm16:0)

? PC 33:2 788.545 778.516

PC O-38:3

[M+HCOO]-

842.6285

842.628 951

GlcCer(d18:0/22:0) ? HexCer d40:0 784.667 830.673 820.644

PC(O-14:0/22:0)

PC(O-16:0/20:0)

PC(O-18:0/18:0)

PC(O-20:0/16:0)

? PC O-36:0 820.644 810.615

PC(20:1/dm18:1)

PC(20:2/dm18:0)

PC(22:2/dm16:0)

? PC 37:2 844.607 834.579

no match 525.338 707 LysoPC(dm16:0) ? LPC 15:0 526.315 516.286

Lipid features reported in negative ion mode

Drogan et al. Clin Chem. 2015 Mar;61(3):487-97

Information provided by Drogan et al.

LIPID MAPS

database search1

Reported

m/z RT [s] Reported lipid species Adduct ion

Appropriate lipid

species

annotation2

Expected m/z values3

Positive ion mode [M+H]+ [M+Na]+ [M+K]+

no match 896.573 943 PC(O-18:0/22:5) ? PC O-40:5 822.637 844.619 860.593

PC(22:4/dm18:0) ? PC 39:4 824.616 846.598 860.557

no match 811.609 957

PC(38:4) ? PC 38:4 810.601 832.583 848.557

PC(36:1) ? PC 36:1 788.616 810.598 826.572

PE(O-18:0/O-18:0)

PE(O-20:0/O-16:0) ? PE dO-36:0 720.627 742.608 758.582

Lipid features reported in positive ion mode

Drogan et al. Clin Chem. 2015 Mar;61(3):487-97

Lipid species annotation

is not correct for most of

the species

Potential biomarkers

are not validated

Standards for validation of

(lipid)biomarkers including

appropriate use of lipid nomenclature

Reply by the authors

Dunn WB, Boeing H. Clin Chem. 2015 Dec;61(12):1544-6.

USE OF DATABASES TO IDENTIFY

LIPID SPECIES

Why is there a need for standardization?

28-Feb-2012, 13:33:46CStk2-01:1

m/z660 680 700 720 740 760 780 800 820 840 860 880 900

%

0

100

PC Fibros 13 (0.401) Sm (SG, 2x0.70); Cm (4:27) Parents of 184ES+ 1.09e7760.5

678.5

703.6

679.5

732.6

704.5

706.5

730.5

758.5

746.6

761.5

786.6

762.6

782.6

788.7

902.7813.6

789.6815.6

903.7

Precursor ion scan m/z 184 of human skin fibroblasts

IS

IS

How to annotate

this peak?

LIPID MAPS Structure Database (LMSD)

http://www.lipidmaps.org/data/structure/LMSDSearch.php?Mode=SetupTextOntologySearch

Search: 745.6&ExactMassOffSet=0.1, Main class PC

LM Database returns 24

chemically defined structures • 15 Diacyl species

• 6 Alkyl species

• 4 Plasmalogens

Analysis usually does not reach

that level of structural resolution

Structural information depends on analysis method

QQQ

Head group scan No differentiation of

PC 33:1 and PC O-34:1

HighRes MS

(PC fraction) Differentiation of

PC 33:1 and PC O-34:1

QQQ

FA scan PC 15:0_18:1

PC 16:0_17:1

PC O-16:0_18:1

…

RULES FOR LIPID SPECIES

ANNOTATION

Why is there a need for standardization?

MS shorthand notation defines a common language

GPC 38:4

PtdCho 18:0/20:4

38:4 PC

PC 38:4

Graz

Regensburg Utrecht

Helsinki Cambridge

Benefits of nomenclature standards

Structural resolution of the analysis is reflected

Easy data exchange

Standards for databases

Meta-analysis of different studies is possible

GPC 38:4

PtdCho 18:0/20:4

38:4 PC

PC 38:4

Are we talking of the same lipid species?

LIPIDOMICS STANDARDS

INITIATIVE (LSI)

https://lipidomics-standards-initiative.org

Lipidomics Standards Initiative (LSI)

Cells - Tissues - Biofluids

Preanalytics

Preservation

Lipid extraction

Recovery – Reproducibility

Addition of IS

Mass spectrometry

Direct - LC

Lipid ion monitoring

Lipid identification

Quantification

Quality control

Reporting standards

Lipocentric databases

Resources

MS MS/MS

LR-MS HR-MS

Shotgun

Imaging

Annotation

Identification

Data deconvolution

Quantification

RPLC

NPLC

HILIC

IS

t

IS

t Standardized

reporting

Consideration of lipid

biochemistry

X

LSI covers main lipidomics approaches

Separation mode defines

NP/HILIC RP

t t

IS IS

Polar selectivity

= separation of lipid classes

Nonpolar selectivity

= separation of lipid species

Quantification Separation of isomers

m/z 746.5694

C41H81O8NP

m/z 746.6058

C42H85O7NP PC 33:1

High resolution MS provides structural resolution

Res. 20,000

PC O-34:1

Res. 40,000 Res. 80,000

LSI – Resources on lipid isomeric overlap

LSI – Resources on lipid isobaric overlap

LSI

https://lipidomics-standards-initiative.org

You are invited

to join and to

drive the wheel!

John A. Bowden

Christer S. Ejsing

Kim Ekroos

Michal Holčapek

Makoto Arita

Masanori Arita

Markus R. Wenk

Todd W. Mitchell

Robert Ahrends Gerhard Liebisch

William J. Griffiths

More information: Kim Ekroos - kim@lipidomicsconsulting.com

Gerhard Liebisch - gerhard.liebisch@ukr.de

& members of the workgroup

Thank you for

your attention