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