Lipidomics Quantitative (MRM) methods, mass spectral libraries for lipids, and considerations for the quantitative analysis of clinical samples

Transcription

1 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. aughey, Ph.D. Johns opkins University School of Medicine Department of Neurology ME 0.0: Lecture, MN, Dec, 0

2 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.

3 ommon functions of lipids Structural components of cellular membranes ell signaling Energy storage Signal transduction Membrane trafficking Morphogenesis Endocrine actions (for example steroid hormones)

4 Lipid lass. Fatty acids (FA). Sphingolipids (SP). Phospholipids (PL). Sterol lipids (ST) 5. Glycerolipids (GL) 6. Prenol lipids (PR) 7. Saccharolipids (SL). Polyketides (PK)

5 Subclassification of lipids Fatty Acids (FA) Fatty Acids and onjugates Eicosanoids Docosanoids ctadecanoids Fatty alcohols Fatty aldehydes Fatty esters Fatty amides Fatty nitriles Fatty ethers ydrocarbons xygenated hydrocarbons Fatty acyl glycosides ther Fatty Acyls Glycerolipids (GL) Monoradylglycerols Diradylglycerols Triradylglycerols Glycosylmonoradylglycerols Glycosyldiradylglycerols ther Glycerolipids Sphingolipids (SP) Sphingoid bases eramides Phosphosphingolipids Phosphonosphingolipids Neutral glycosphingolipids Acidic glycosphingolipids Basic glycosphingolipids Amphoteric glycosphingolipids Arsenosphingolipids ther Sphingolipids Glycerophospholipids (GP) Glycerophosphocholines (P) Glycerophosphoethanolamines (PE) Glycerophosphoserines (PS) Glycerophosphoinositols (PI) Glycerophosphoglycerols (PG) Glycerophosphoglycerophosphates Glycerophosphoinositol monophosphates Glycerophosphoinositol bisphosphates Glycerophosphoinositol trisphosphates Glycerophosphates Glyceropyrophosphates Glycerophosphoglycerophosphoglycerols DP-Glycerols Glycosylglycerophospholipids Glycerophosphoinositolglycans Glycerophosphonocholines Glycerophosphonoethanolamines Di-glycerol tetraether phospholipids Glycerol-nonitol tetraether phospholipids xidized glycerophospholipids ther Glycerophospholipids Sterol Lipids (ST) Sterols Steroids Secosteroids Bile acids and derivatives Steroid conjugates ther Sterol lipids Prenol Lipids (PR) Isoprenoids Quinones and hydroquinones Polyprenols opanoids ther Prenol lipids Saccharolipids (SL) Acylaminosugars Acylaminosugar glycans Acyltrehaloses Acyltrehalose glycans ther acyl sugars ther Saccharolipids Polyketides (PK) Linear polyketides alogenated acetogenins Annonaceae acetogenins Macrolides and lactone polyketides Ansamycins and related polyketides Polyenes Linear tetracyclines Angucyclines Polyether polyketides Aflatoxins and related substances ytochalasins Flavonoids Aromatic polyketides Non-ribosomal peptide/polyketide hybrids ther Polyketides

6 Fatty acid A long hydrocarbon (carbon and hydrogen) chain with a carboxyl (acid) group ydrocarbon chain arboxylic acid Fatty acid (R-) R or

7 Fatty acid species Saturated fatty acids (contain - single bonds) Palmitic acid Unsaturated fatty acids (contain = double bonds) Palmitoleic acid

8 Fatty Acids (FA) Saturated FAs Unsaturated FAs Palmitic acid (6:0) [exadecanoic acid] Palmitoleic acid (6:) [exadecaenoic acid ] Stearic acid (:0) [ctadecanoic acid] leic acid (:) [is 9-octadecenoic acid] Arachidic acid (0:0) [Eicosanoic acid] Gondoic acid (0:) [Eicosenoic acid]

9 ω fatty Acids (FA) ω α mega- fatty acid (Linolenic acid, :) ω 5 6 α mega-6 fatty acid (Linoleic acid, :) ω α mega-9 fatty acid (lic acid, :)

10 mega (ω)fatty acids mega-9 fatty acids, mono- and polyunsaturated ctadecaenoic acid (leic acid) : Eicosenoic acid 0: Docosaenoic acid : Tetracosaenoic acid : exacosaenoic acid 6: Eicosatrienoic acid 0: mega-6 fatty acids exadecadienoic acid 6: ctadecadienoic acid (Linolic acid) : Eicosadienoic acid 0: Docosadienoic acid : Tetracosadienoic acid : exacosadienoic acid 6: mega- fatty acids exadecatrienoic acid 6: ctadecatrienoic acid (Linolenic acid) : Docosatrienoic acid : Tetracosatrienoic acid : exacosatrienoic acid 6: ---ther fatty acids

11 Fatty Acids (Eicosanoids) Arachidoic acid (0:) [5Z, Z, Z, Z- eicosatetraenoic acid] 5 (6)-EET [(±)5(6)-epoxy-Z, Z,Z- eicosatrienoic acid] Docosahexaenoic Acid (DA) Z, 7Z, 0Z, Z, 6Z, 9Z- docosahexaenoic acid (+) 5-ETE [(±)5- hydroxy- 6E, Z, Z, Z- eicosatetraenoic acid]

12 Nomenclature and structural difference of prostaglandins (PGs) 0 9 R 6 5 R Prostanoic Acid R R R R R R R R R R R R PGA PGB PG PGD PGE PGFα R R R R R R R R R R PGG, PGI PGJ PGK TXA TXB R R

13 lasses of prostaglandins (PGs) st series series series PGs A, B,, D, E, F, G,, etc PGs A, B,, D, E, F, G,, etc PGs A, B,, D, E, F, G,, etc

14 Examples of prostaglandins (PGs) PGD PGE Leukotriene B -iso PGD

15 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

16 Glycerophospholipids Fatty acyl chains ---R N PA (Phosphatidic acid) PE (Phosphatidylethanolamine) - P X ---R --P- X Glycerol precursor _ N N P (Phosphatidylcholine) PS (Phosphatidylserine) PG (Phosphatidylglycerol) PI (Phosphatidylinositol)

17 Phospholipid structure + N + N - + N + N P - P - P - P - FATTY AID AID FATTY AID AID FATTY AID AID FATTY AID AID ERAMIDE Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine Sphingomyelin

18 Function of phospholipids Phosphatidic acid Glycerophosphoethanolamine Glycerophosphocholine Signaling, intermediates Membrane bilayer Membrane bilayer Glycerophosphoserine Membrane bilayer Glycerophosphoinositol Glyverophosphoglycerol ardiolipin Membrane bilayer Membrane bilayer Mitochondria bilayer

19 Sphingolipids - - -R -N--R - eramide Sphingomyelin --X Glucosylceramide Lactosylceramide N X Ganglioside GM

20 Sphingolipid nomenclature and structures d: Sphingosine N G M Ganglioside d:/6:0 eramide N d:/:0 Sphingomyelins N P + N

21 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

22 Glycerolipids (GL) R= Saturated or unsaturated fatty acids Glycerol R Acyl group R R R R R R Monoglycerols Diglycerols Triglycerols -and other glycerolipids R R R

23 Functions of glycerol Triglycerides Energy storage Diacylglycerols Signaling, intermediates Monoetherdiacylglycerols Intermediates

24 Sterols holesterol (cholest-5-en-b-ol) S-hydroxycholesterol (cholest-5-en-β,s-diol) -hydroxycholesterol (cholest-5-en-β,-diol) 7-hydroxycholesterol (cholest-5-en-β,6-diol) -and other sterol lipids

25 Functions of sterols Regulatory functions ellular signaling Integral building block of cell membranes Modulate fluidity

26 Prenol lipids (PR) Saccharolipid Kdo-Lipid A - P - P - 5 isoprenoids: isopentenyl pyrophosphate (-methylbut--nyl pyrophosphate) P N N P Polyketides (PK) N N Megalomicin A

27 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

28 Techniques used for the analysis of lipidomic profiles igh performance liquid chromatography (PL) igh-performance thin-layer chromatography (PTL) Spectroscopic (Nuclear magnetic resonance, NMR) Mass spectrometric methods (MS) and combinations of the above techniques ie PL/MS

29 Mass spectrometry methods Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TF-MS) Imaging mass spectrometry (IMS). An application of MALDI-TF MS Electrospray ionization (ESI) Atmospheric pressure chemical ionization (API) Most widely used methods in the lipid field are ESI and API

30 L/ESI/MS/MS L ESI MS MS Detector Turbo-Spray Molecular ions Fragmented ions olumn Auto sampler Q mass analyzer Electron impact ionizer (ID) Q collision cell Q mass analyzer L pump Solvent A Solvent B L or PL L = Liquid chromatography or igh pressure liquid chromatography (PL) ESI = Electrospray ionization MS and MS = Mass spectrometry analyzers ID = ollision induced dissociation via neutral gas (N /Ar) olumn=stationary phase Solvents A and B=Mobile phases

31 lass of lipid Fatty acyls (FA) Sphingolipids (SP) Phospholipids (PL) Sterol lipids (ST) Quadruple charge (mode +ve or -ve) -ve +ve and ve +ve and ve +ve

32 General methods for fragmentation Electron capture dissociation (ED) Multiply-charged cations capture electrons, inducing odd-electron fragmentation Infrared multiphoton dissociation (IRMPD) Bond cleavage by using infrared laser (hν) ollision induced dissociation (ID) In the presence of an electric field to accelerate precursor ions and induce weakest bond cleavage via collision with neutral gas molecules (N or Ar)

33 Steps in L/ESI/MS/MS analysis of lipids A) Reference and internal standards (IS) B) Sample preparation ) Instrumentation D) Method (MRM) development E) Validation F) Data analysis

34 A) Reference and internal standards Selection of appropriate standards B) Sample preparation By liquid liquid extraction or Solid phase extraction (SPE) ) Instrumentation Pumps Autosampler olumn Mass spectrometer

35 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 ptimization of mass spec parameters. ompound dependent. Source

36 E) Validation Sensitivity Slope of the graph for the sample concentration versus the measured signal. Evaluated by determining the limit of detection (LD) and limit of quantification (LQ) LD Signal-to-noise ratio of at least and Lower limit of quantification (LLQ) 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 0) Upper limit of quantification (ULQ) ighest amount of analyte in a sample which can be quantitatively determined with pre-defined precision and accuracy.

37 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 (00 + 0%) alibration 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)

38 Precision (intra-assay and inter-assay precision) The closeness of repeated individual measures of analyte which can expressed as the coefficient of variation (V). 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

39 Fragmentation of ceramide N N m/z 6

40 MS/MS spectrum.6e7 6. Intensity (cps).e7.e7.e7.0e7.6e7 N.e7.0e6.0e m/z (amu)

41 eramides.6 Internal standard (IS).5 :0 6:0 9.5e5.5e5 Total ion chromatogram (TI) :0.7 :0 7.5e5 :0 Intensity (cps) :0 :0 :0 6: Time (min) Intensity (cps) 6.5e5 5.5e5.5e5.5e5.5e5.5e5 5.0e 0.0 :0 6:0 0:0 :0 6: Time (min)

42 alibration curves for ceramides