Guidelines for Lipidomics Analysis and Reporting the ... · Universitätsklinikum Regensburg...
Transcript of Guidelines for Lipidomics Analysis and Reporting the ... · Universitätsklinikum Regensburg...
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 - [email protected]
Gerhard Liebisch - [email protected]
& members of the workgroup
Thank you for
your attention