lipidomics quantitative (mrm) methods, mass …...lipidomics quantitative (mrm) methods, mass...
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Lipidomics Quantitative (MRM) methods, mass spectral libraries
for lipids, and considerations for the quantitative analysis of clinical samples
Veera Venkata Ratnam Bandaru, Ph.D. Norman J. Haughey, Ph.D.
Johns Hopkins University School of Medicine
Department of Neurology
ME 330.804: Lecture 11, MON 3, Dec, 2012
Lipid (definition) Any of a class of organic compounds that are fatty acids or their derivatives and are soluble in organic (or non polar) solvents but insoluble in water (polar solvents). Examples: Natural oils, waxes, and steroids.
Common functions of lipids
• Structural components of cellular membranes • Cell signaling • Energy storage • Signal transduction • Membrane trafficking • Morphogenesis • Endocrine actions (for example steroid hormones)
1. Fatty acids (FA) 2. Sphingolipids (SP) 3. Phospholipids (PL) 4. Sterol lipids (ST) 5. Glycerolipids (GL) 6. Prenol lipids (PR) 7. Saccharolipids (SL) 8. Polyketides (PK)
Lipid Class
Fatty Acids (FA) Fatty Acids and Conjugates Eicosanoids Docosanoids Octadecanoids Fatty alcohols Fatty aldehydes Fatty esters Fatty amides Fatty nitriles Fatty ethers Hydrocarbons Oxygenated hydrocarbons Fatty acyl glycosides Other Fatty Acyls Glycerolipids (GL) Monoradylglycerols Diradylglycerols Triradylglycerols Glycosylmonoradylglycerols Glycosyldiradylglycerols Other Glycerolipids Sphingolipids (SP) Sphingoid bases Ceramides Phosphosphingolipids Phosphonosphingolipids Neutral glycosphingolipids Acidic glycosphingolipids Basic glycosphingolipids Amphoteric glycosphingolipids
Arsenosphingolipids Other Sphingolipids Glycerophospholipids (GP) Glycerophosphocholines (PC) Glycerophosphoethanolamines (PE) Glycerophosphoserines (PS) Glycerophosphoinositols (PI) Glycerophosphoglycerols (PG) Glycerophosphoglycerophosphates Glycerophosphoinositol monophosphates Glycerophosphoinositol bisphosphates Glycerophosphoinositol trisphosphates Glycerophosphates Glyceropyrophosphates Glycerophosphoglycerophosphoglycerols CDP-Glycerols Glycosylglycerophospholipids Glycerophosphoinositolglycans Glycerophosphonocholines Glycerophosphonoethanolamines Di-glycerol tetraether phospholipids Glycerol-nonitol tetraether phospholipids Oxidized glycerophospholipids Other Glycerophospholipids Sterol Lipids (ST) Sterols Steroids Secosteroids Bile acids and derivatives Steroid conjugates Other Sterol lipids
Prenol Lipids (PR) Isoprenoids Quinones and hydroquinones Polyprenols Hopanoids Other Prenol lipids Saccharolipids (SL) Acylaminosugars Acylaminosugar glycans Acyltrehaloses Acyltrehalose glycans Other acyl sugars Other Saccharolipids Polyketides (PK) Linear polyketides Halogenated acetogenins Annonaceae acetogenins Macrolides and lactone polyketides Ansamycins and related polyketides Polyenes Linear tetracyclines Angucyclines Polyether polyketides Aflatoxins and related substances Cytochalasins Flavonoids Aromatic polyketides Non-ribosomal peptide/polyketide hybrids Other Polyketides
Subclassification of lipids
Fatty acid
A long hydrocarbon (carbon and hydrogen) chain with a carboxyl (acid) group
H C C C
H
H
H
H H
H
Hydrocarbon chain Carboxylic acid
R H C C C C
H
H
H
H H
H
O
OHC
O
OH
Fatty acid (R-COOH)
or
HOH
O
Fatty acid species
• Saturated fatty acids (contain C-C single bonds)
Palmitic acid
• Unsaturated fatty acids (contain C=C double bonds)
OH
O
Palmitoleic acid OH
O
Fatty Acids (FA)
OH
O
12
34
56
78
910
1112
1314
1516
OH
O
12
34
56
78
910
1112
1314
1516
1718
OH
O
12
34
56
78
910
1112
1314
1516
1718
1920
OH
O
12
34
56
78910
1112
1314
1516
OH
O
12
34
56
78910
1112
1314
1516
1718
Saturated FAs Unsaturated FAs
Palmitic acid (16:0) [Hexadecanoic acid]
Stearic acid (18:0) [Octadecanoic acid]
Arachidic acid (20:0) [Eicosanoic acid]
Oleic acid (18:1) [Cis 9-octadecenoic acid]
Gondoic acid (20:1) [Eicosenoic acid]
Palmitoleic acid (C16:1) [Hexadecaenoic acid ]
OH
O
12
34
56
78
910
111213
1415
16
1718
1920
1 3ωOH
O
α
1 3 5ω αOH
O
7 9
Omega-9 fatty acid (Olic acid, C18:1)
Omega-3 fatty acid (Linolenic acid, C18:3)
Omega-6 fatty acid (Linoleic acid, C18:2)
1 3 5 6ωOH
O
α
ω-fatty Acids (FA)
Omega (ω)fatty acids Omega-9 fatty acids, mono- and polyunsaturated Octadecaenoic acid (Oleic acid) C18:1 Eicosenoic acid C20:1 Docosaenoic acid C22:1 Tetracosaenoic acid C24:1 Hexacosaenoic acid C26:1 Eicosatrienoic acid C20:3 Omega-6 fatty acids Hexadecadienoic acid C16:2 Octadecadienoic acid (Linolic acid) C18:2 Eicosadienoic acid C20:2 Docosadienoic acid C22:2 Tetracosadienoic acid C24:2 Hexacosadienoic acid C26:2 Omega-3 fatty acids Hexadecatrienoic acid C16:3 Octadecatrienoic acid (Linolenic acid) C18:3 Docosatrienoic acid C22:3 Tetracosatrienoic acid C24:3 Hexacosatrienoic acid C26:3
---Other fatty acids
Fatty Acids (Eicosanoids)
5 (6)-EET [(±)5(6)-epoxy-8Z,11Z,14Z- eicosatrienoic acid]
(+) 5-HETE [(±)5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid]
COOH
OH
12
34
567
89
10
11 12 1314 15
1617
1819
20
COOH
O1
23
456
789
10
11 1213
14 1516
1718
1920
Docosahexaenoic Acid (DHA) 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid
COOH1
23
456
789
10
11 1213
14 1516
1718
1920
Arachidoic acid (C20:4) [5Z,8Z,11Z,14Z-eicosatetraenoic acid]
COOH
12
345
678
91011
12
13 1415
16 1718
19 2021
22
Nomenclature and structural difference of prostaglandins (PGs)
O
R1
R2
O
R1
R2
OH
O
R1
R2
O
OH
R1
R2
OH
OH
R1
R2
O
R1
R2
O
R1
R2
O
O
R1
R2
O
OH
R1
R2
O
OR1
R2 OOH
OH
R1
R2O
O
R1
R2
R1
R2
Prostanoic Acid
COOH3
45
689
10
11 1213
1415
1617
1819
1
PGA PGB PGC PGD PGE PGFα
PGG,H PGI PGJ PGK TXA TXB
20
Classes of prostaglandins (PGs)
PGs A1, B1, C1, D1, E1, F1, G1, H1, etc…
PGs A2, B2, C2, D2, E2, F2, G2, H2, etc…
PGs A3, B3, C3, D3, E3, F3, G3, H3, etc…
OHOH
234678
1011
13 1415
16 17 18 19
1COOH
5
12
2346789
10
1113 14
1516 17 18 19
1
20
COOH5
12
2 series
COOH23456789
10
11 12 13 14 15 16 17 18 19
1
20
1st series 3 series
20
COOH
O
OH
OHPGD2
COOH
O
HOOH
PGE2
COOHOH OH
Leukotriene B4
COOH
OH
OOH 8-iso PGD1
Examples of prostaglandins (PGs)
Fatty acyls of arachidonic acid regulate a wide variety of cellular functions
• Inflammation • Neurodegeneration • Neurotransmitter release • Membrane trafficking • Growth and differentiation • Membrane remodeling • Long-term potentiation • Membrane fusion events
Glycerophospholipids
Fatty acyl chains
Glycerol precursor
O _
1 H2C -O-C-R 1
3 H2C -O-P-O X
2 HC-O-C-R 2
O
O
O
PI (Phosphatidylinositol) O H
O H H O H O O H
- H
- C H 2 - C H 2 - N C H 3 C H 3 C H 3
- C H 2 - C H 2 - N H 2
- C H 2 - C H 2 - + N H 3 C O O -
- C H 2 - C H - C H 3 O H O H
PA (Phosphatidic acid)
PE (Phosphatidylethanolamine)
PC (Phosphatidylcholine)
PS (Phosphatidylserine)
PG (Phosphatidylglycerol) O
OO
OPO
X
O-O
O
1
2
3
Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine Sphingomyelin
Phospholipid structure
CH2
+NH3
CH2
O
PO O-
CH2CHCH2
O O
C O C O
FAT
TY
AC
ID
AC
ID
H C
+NH3
CH2
O
PO O-
CH2CHCH2
O O
C O C O
COO-
FAT
TY
AC
ID
AC
ID
CH3CH3CH3
CH2
+N
CH2
O
PO O-
CH2CHCH2
O O
C O C O
FAT
TY
AC
ID
AC
ID
CH3CH3CH3
CH2
+N
CH2
O
PO O-
CE
RA
MID
E
FAT
TY
AC
ID
AC
ID
Function of phospholipids • Phosphatidic acid Signaling, intermediates
• Glycerophosphoethanolamine Membrane bilayer
• Glycerophosphocholine Membrane bilayer
• Glycerophosphoserine Membrane bilayer
• Glycerophosphoinositol Membrane bilayer
• Glyverophosphoglycerol Membrane bilayer
• Cardiolipin Mitochondria bilayer
- H 3 HO-C H-CH -R 1
1 H2C -O-X
2 HC-N-C-R 2
O H 2 Ceramide
Sphingomyelin
Glucosylceramide
Lactosylceramide
Ganglioside GM2
Sphingolipids
CH2
OH
HN
O
OX
1
2
3
GM1
d18:1/16:0 Ceramide
d18:1/18:0 Sphingomyelins
OPO
OH
O
+NCH3
CH3CH3
OH
NH
O
12
3
4
5
6
7
89
1011
1213
1415
1617
18
12
34
56
78
910
1112
1314
1516
1718
OH
OH
NH2
12
3
4
56
7
89
10
11
12
13
14
15
16
1718
d18:1 Sphingosine
Sphingolipid nomenclature and structures
H2C
OH
NH
O
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
12
34
56
78
910
1112
1314
1516
1718
OH
Ganglioside
Sphingolipids regulate a wide variety of biological processes
• Biophysical properties of membranes • Lipid-protein interactions • Protein scaffolding and post-translational modifications
Brain is especially enriched in sphingolipids • Neutrotropic signaling • Neural cell adhesion and migration • Synaptic transmission • Axonal guidance • Neuron-glial interactions
Glycerolipids (GL)
R= Saturated or unsaturated fatty acids
C
C
C
OH
OH
OH
H
H
H
H
H
Glycerol
CR
O
H
Acyl group
C
C
C
H
H
O
O
O
H
C
C
R1
R2
O
O
H
C OH
O
H
C
C
C
H
H
O
O
O
H
C
C
R1
HO
O
O
H
C OH
O
H
C
C
C
H
H
O
O
O
H
C
C
R1
R2
O
O
H
C R3
O
H
Monoglycerols Diglycerols Triglycerols
-and other glycerolipids
C
C
C
H
H
O
O
O
H
C
C
R
R
O
O
H
C R
O
H
1
2
3
Functions of glycerol
• Triglycerides Energy storage
• Diacylglycerols Signaling, intermediates
• Monoetherdiacylglycerols Intermediates
Cholesterol (cholest-5-en-3b-ol)
Sterols
HO
12
34 5
6
7
8
9
10
11
12
13
1415
16
1718
19
20
21 22
23
24
25
26
27HO
HO
12
34 5
6
7
8
9
10
11
12
13
1415
16
1718
19
20
21 22
23
24
25
26
27
OH
HO
12
34 5
6
7
8
9
10
11
12
13
1415
16
1718
19
20
21 22
23
24
25
26
27
OH
27-hydroxycholesterol (cholest-5-en-3β,26-diol)
24S-hydroxycholesterol (cholest-5-en-3β,24S-diol)
22-hydroxycholesterol (cholest-5-en-3β,22-diol)
-and other sterol lipids
HO
12
34 5
6
7
8
9
10
11
12
13
1415
16
1718
19
20
21 22
23
24
25
26
27
Functions of sterols
• Regulatory functions • Cellular signaling • Integral building block of cell membranes • Modulate fluidity
OP
OP
O-
OO
-O-O
Prenol lipids (PR) Saccharolipid Kdo2-Lipid A
O
O
OH
O
OOH
OHHO
O
OH
O
O
OP
HOHO
O
OHOH
HO
HO
O
O
OHO
O
OP
OHHO
O
NH
O
HO
O
HO
NH
O
O
O
O
O
O
Polyketides (PK) O
OH
N
O
O
O
O
HO
O
O
ON
O
HO
OH
OH
OH
C5 isoprenoids: isopentenyl pyrophosphate
(3-methylbut-3-3nyl pyrophosphate)
Megalomicin A
Functions of prenol
• Anti-oxidants and as precursors of vitamin A Function of saccharolipids
• Membrane bilayer Functions of polyketides
• Messengers in cell-to-cell communication
Techniques used for the analysis of lipidomic profiles • High performance liquid chromatography (HPLC) • High-performance thin-layer chromatography (HPTLC) • Spectroscopic (Nuclear magnetic resonance, NMR) • Mass spectrometric methods (MS) …and combinations of the above techniques – ie HPLC/MS
Mass spectrometry methods
• Matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS) • Imaging mass spectrometry (IMS). An application of MALDI-TOF MS
• Electrospray ionization (ESI) • Atmospheric pressure chemical ionization (APCI) Most widely used methods in the lipid field are ESI and APCI
LC/ESI/MS/MS
LC = Liquid chromatography or High pressure liquid chromatography (HPLC) ESI = Electrospray ionization MS1 and MS2 = Mass spectrometry analyzers CID = Collision induced dissociation via neutral gas (N2/Ar) Column=Stationary phase Solvents A and B=Mobile phases
ESI
Detector
Electron impact ionizer (CID)
Molecular ions
Fragmented ions
Q3 mass analyzer
Q1 mass analyzer
Q2 collision cell
LC
Turbo-Spray
MS1 MS2
LC pump
Auto sampler
Column
Solvent A Solvent B
LC or HPLC
Class of lipid
Quadruple charge (mode +ve or -ve)
Fatty acyls (FA) -ve Sphingolipids (SP) +ve and –ve Phospholipids (PL) +ve and –ve Sterol lipids (ST) +ve
General methods for fragmentation
• Electron capture dissociation (ECD)
Multiply-charged cations capture electrons, inducing odd-electron fragmentation
• Infrared multiphoton dissociation (IRMPD)
Bond cleavage by using infrared laser (hν) • Collision induced dissociation (CID)
In the presence of an electric field to accelerate precursor ions and induce weakest bond cleavage via collision with neutral gas molecules (N2 or Ar)
Steps in LC/ESI/MS/MS analysis of lipids
A) Reference and internal standards (IS) B) Sample preparation C) Instrumentation D) Method (MRM) development E) Validation F) Data analysis
A) Reference and internal standards • Selection of appropriate standards
B) Sample preparation
• By liquid liquid extraction or • Solid phase extraction (SPE)
C) Instrumentation
• Pumps • Autosampler
• Column • Mass spectrometer
D) Method (MRM) development
• Selection of quadrupole charge (+ve or –ve mode)
• Molecular ion identification
• Fragmented ions identification
• Selection of mobile phases
• Selection of stationary phase
• Optimization of mass spec parameters
1. Compound dependent 2. Source
E) Validation
Sensitivity • Slope of the graph for the sample concentration versus the measured signal. Evaluated by determining the limit of detection (LOD) and limit of quantification (LOQ)
LOD • Signal-to-noise ratio of at least 3 and
Lower limit of quantification (LLOQ)
• Lowest amount of analyte in a sample which can be quantified reliably, with an acceptable accuracy and precision
(Signal-to-noise ratio of at least 10) Upper limit of quantification (ULOQ)
• Highest amount of analyte in a sample which can be quantitatively determined with pre-defined precision and accuracy.
Selectivity • Evaluated by differentiate the analyte of interest and IS from endogenous components in the matrix or other components in the sample.
Stability
• Determined by freeze-thaw cycles Accuracy
• Evaluated by comparing the percentage targeted analyte concentration to expected concentration (100 + 20%)
Calibration curve, linearity, and dynamic range • Determined with a constant mass (IS) and variable concentrations of reference standards mixture. (The instrument response and calibration curves were linear within the selected dynamic range)
Precision (intra-assay and inter-assay precision)
• The closeness of repeated individual measures of analyte which can expressed as the coefficient of variation (CV).
Recovery and matrix effect
• Evaluated by comparing analyte peak areas of extracted samples with the analyte peak areas of neat samples prepared in mobile phase at an equivalent concentration.
F) Data analysis
Quantification
Fragmentation of ceramide
m/z 264
OH
NH
O
OH
OH
NH
O
OH
100 180 260 340 420 500
4.0e6
8.0e6
1.2e7
1.6e7
2.0e7
2.4e7
2.8e7
3.2e7
3.6e7
264.4
482.9
m/z (amu)
Inte
nsity
(cps
) OH
NH
O
OH
MS/MS spectrum
1.63
2.52
4.04
5.27
6.90
Time (min) 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5
8.93
Inte
nsity
(cps
)
C12:0
C16:0
C20:0
C22:0
C24:0
C26:0
3.17 C18:0
Internal standard (IS)
0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 Time (min)
0.0 5.0e4
1.5e5
2.5e5
3.5e5
4.5e5
5.5e5
6.5e5
7.5e5
8.5e5
9.5e5
Inte
nsity
(cps
)
Ceramides
C12:0
Total ion chromatogram (TIC)
C16:0
C18:0
C20:0
C22:0
C24:0
C26:0
Calibration curves for ceramides