Mass Spectrometry based metabolomics Metabolomics- A realm of small molecules (<1000 Da) Jeevan Prasain, PhD
What is metabolomics? Identification and quantification of the complete set of metabolites in a biological system Quantitative global analysis of metabolites from cells, tissues and fluids Quantitative measurement of the dynamic metabolite response of living systems to pathophysiological stimuli or genetic modification
Workflow for metabolome analysis Sample collection Treatment or Diseased group Control group Urine, plasma, tissue etc. Sample preparation Sample analysis Data export Internal standard spiking for quantitative analysis Extraction (liquid-liquid, ppt with or without hydrolysis) LC-MS and LC-MS/MS analysis Q1 scan using HPLC or UPLC/-TFMS +ve/-ve ion mode- accurate mass measurement MS/MS experiments using a hybrid instrument Q-Trap Data analysis Multivariat analysis e.g. PCA Marker identification
Metabolomics in the context of other omics Genome Transcriptome Proteome Metabolome What might be happening in a cell Snapshot of the entire physiology Lipidomics Lipids are important- as a membrane bilayer - provides hydrophobic environment for protein function - reservoir of energy - signaling molecules Lipidomics can perhaps best be defined as a comprehensive analysis of lipids on the systems-level scale together with their interacting factors
utlines Brief introduction to lipidomics Analytical methodology: MS/MS structure elucidation of phospholipids Phospholipid analysis in lean and ob/ob mice by mass spectrometry MS/MS analysis of eicosanoids
Structures of different lipids classes H CH 2 H Ceramide R NH
Structures of main phospholipids Sn-1 Sn-2 Sn-3 Phosphatidylethanolamine (PE) Y= NH 2 Phosphatidylcholine (PC) Y= N(CH 3 ) 3 Phosphatidylglycerol (PG) Y= H H Phosphatidylserine (PS) Y= H NH 2 Phosphatidylinositol (PI) Y= H H H H P H P H Cardiolipin (diphosphatidylglycerol) H H
How phospholipids are synthesized? Choline Phosphocholine Cytidine diphosphocholine 1,2-diacylglycerol Phosphatidylcholine Activation of phospholipase A2 Lysophosphatidylcholine (LPC) + Free fatty acid Pro-inflammatory property Component of oxidized LPL Phosphatidylethanolamine Hydrolysis by phospholipase A2 Lysophosphatidylethanolamine (LPE)
Extraction of lipids by Bligh/Dyer method To a homogenized sample (1 ml containing internal standards) add methanol (2.5 ml) and chloroform (1.25 ml), sonicate by 4-5 bursts and added 1.0 ml water and 1.25 ml chloroform additionally and vigorously shaken. Centrifuge (1,000 x g) for 2 min and separate the chloroform layer (bottom layer) and repeat the process twice. Combine the chloroform soluble phase and evaporate to dryness and stored at -20 o Cuntill analysis.
Shotgun lipidomics: intrasource separation of lipids for quantitative lipidomics The ionization efficiency of an analyte greatly depends on the electrical propensity of an individual analyte in its own microenvironment to lose or gain a charge Source: Gross and Han,, 2004
Which ionization mode for which phospholipids? Positive ion mode Negative ion mode PC LPC PE LPE SM PS PE PA PI PI PG PIPs PC = phosphatidylcholine PA = phosphatic acid PE = phosphatidylethanolamine PS = phosphatidylserine PG = phosphatidylglycerol PI = phosphatidylinositol PIP = PI monophosphate SM = sphingomyelin LPE = lysope
Increasing metabolite coverage using +ve and ve ion mode No arachidonic Acid in +ve ion mode Representative Q1 scans of a methanolic extract of human blood serum Source: Nordstrom et al. Analytical Chemistry, 2007
Application of shotgun lipidomics: intra-source separation of lipids Source: Gross and Han, methods in Enzymology, 2007
Total scan of metabolites (Q1 SCAN + ion mode) for a plasma sample obtained from lean mouse [A]; ob/ob mouse Total metabolomics 100 496.361 518.345 [A] 758.599 869 542.347 544.367 786.632 % 546.380 806.604 810.628 566.351 568.366 811.636 590.351 812.644 494.352 591.359 756.584 835.632 0 703.605 400 475 550 625 700 775 850 900 m/z 100 496.335 518.318 544.339 [B] 758.553 760.570782.552 1.52e3 % 546.352 566.322 806.556 810.592 811.599 812.608 835.594 568.337 494.326 590.321 602.288 732.558 0 400 475 550 625 700 775 850 m/z
Total scan of metabolites (Q1 SCAN -ve ion mode) for a plasma sample obtained from lean mouse [A]; ob/ob mouse 100 480.425 20.3 % 504.432 508.464 558.466 478.419 588.418 794.751 885.817 646.397 674.430 820.780 0 732.383 830.818 886.779 400 475 550 625 700 775 850 900 m/z 100 480.415 52.5 % 504.431 506.445540.459 508.463 564.474588.456 0 794.760816.775 400 475 550 625 700 775 850 900 m/z
Tandem mass spectrometry (MS/MS) of phospholipids H R 1 P R 2-141 NH 2 Phosphatidylethanolamine m/z 184 185 Phosphatidylserine
MS/MS in negative ion mode of phospholipids provide information about the fatty acyl chain
ESI-MS/MS analyses of various lipids Source: Gross and Han,, 2004
Focused lipidomics A. Flow injection (ESI-MS/MS) -Precursor ion scanning at m/z 184-choline-containing phospholipids +ve ion mode - Neutral scanning of 141, 185, 189, and 277 u used for PE, PS, phosphatidylglycerol (PG), and phosphatidylinositol (PI), respectively - precursor ion scanning at m/z 153 and 241 in ve ion mode-glycerol-containing phospholipids and inositol-containing phospholipids, respectively
+Prec (184.00): Exp 1, 2.764 to 30.604 min from Sample 11 (Lean 53 NK) of ZhangSET1.wiff (Turbo Spray), Centroided 2.7e6 759.1 761.1 783.0 787.1 807.1 Intensity, cps 496.9 525.0 811.1 762.1 788.2 400 460 520 580 640 700 760 820 880 835.1 704.1 781.1 814.1 544.8 733.0 836.1 494.8 546.9 568.8 702.0 767.9 795.4 690.0 805.0 +Prec (184.00): Exp 1, 2.764 to 30.603 min from Sample 16 (b 27 NK) of ZhangSET1.wiff (Turbo Spray), Centroided 3.1e6 807.1 761.1783.0 759.1 Intensity, cps 787.0 496.9 808.1 525.0 400 460 520 580 640 700 760 820 880 811.1 522.9 520.8 704.1 789.1 757.0 781.1 814.2 544.7 733.0 805.0 494.8 546.9 568.8 702.0 791.3 747.0 769.3 797.0 817.2 837.1 835.1
+NL (141.00): Exp 2, 2.278 to 33.485 min from Sample 11 (Lean 53 NK) of ZhangSET1.wiff (Turbo Spray), Centroided Intensity, cps 6239 [M+H]+, m/z 452 = 16:1 lysoetn [M+H]+, m/z 454 = 16:0 lysoetn [M+H]+, m/z 478 = 18:2a lysoetn [M+H]+, m/z 482 = 18:0 lysoetn [M+H]+, m/z 502 = 20:4a lysoetn [M+H]+, m/z 526 =? 400 460 520 580 640 700 760 820 880 744.9 764.9 769.0 778.8 478.3 502.6 745.8 454.5 482.5 526.5 716.9 740.9 750.8 779.8 792.8 483.5 751.8 636.8 729.1 548.5 703.0 738.9 762.8 430.1 452.4 476.5 508.4 528.7 583.7600.5606.6 619.5 661.7690.8 732.2 797.0 810.8 892.5 +NL (141.00): Exp 2, 2.278 to 33.485 min from Sample 15 (b 26 NK) of ZhangSET1.wiff (Turbo Spray), Centroided Intensity, cps 3400 454.5 482.5 [M+H]+, m/z 716 = 34a:2 GPEtn [M+H]+, m/z 738 = 36a:5 GPEtn [M+H]+, m/z 740 = 36a:4 GPEtn [M+H]+, m/z 762 = 38e:0 GPEtn [M+H]+, m/z 764 = 38a:6 GPEtn [M+H]+, m/z 768 = 38a:4 GPEtn [M+H]+, m/z 778 = 40p:5/40e:6 GPEtn [M+H]+, m/z 792 = 40a:6 GPEtn [M+H]+, m/z 794 = 40a:5 GPEtn 740.9 769.0 764.9 400 460 520 580 640 700 760 820 880 900 769.9 480.5 502.5 716.8 478.5483.5 526.5 792.9 778.9 762.7 659.3 738.8 794.9 430.0 528.6 426.1 452.4 524.4 548.3 581.7 600.4 636.8 663.6 687.4 715.9 752.8 786.7795.9812.9 836.9 857.9 898.8
TF MSMS 494.35ES+ MSMS fragmentation of m/z 496 obtained from plasma of ob/ob mouse supplemented with kudzu H P H m/z 125 100 313.285 478.348 100 184.080 % 104.113 125 % -59 258.118 419.277-18 496.357 239.247 311.266 0 283.283 417.264 476.331 220 280 340 400 460 520 m/z 86.104 478.348 0 75 150 225 300 375 450 525 m/z
MS/MS of m/z 480 [M-15]- from a ob/ob no kudzu supplemented plasma sample TF MSMS 480.40ES- 100 255.267 + m/z 480, [M-15]- % C16:0 0 224.102 242.116 256.275 220 280 340 400 460 m/z
Several isomeric compounds exits and unambiguous identification is a challenge sn-1 sn-2 H P H N m/z 524 LysoPC P H N m/z 524 LysoPC P H N m/z 524 Platelet activating factor
Lithiated adducts of phosphocholine provide more structural information in their MS/MS spectra Source: Hsu et al. J. Am Soc. Mass Spectrom, 1998
Relative abundances of product ion can be used to distinguish positional isomers of lithiated phospholipids Source: Hsu et al. J. Am Soc. Mass Spectrom, 1998
A 2D ESI mass spectrometric finger print for TG molecules Source: Han and Gross, 2004
MS/MS analysis of eicosanoids Eicosanoids, meaning 20 derived from a 20-carbon acid, arachidonic acid -Important lipid mediators and elicit potent effects in various biological systems mediated through specific protein receptors
Structural representation PG based on ring features R = aliphatic chain
ESI-MS/MS of the [M-H]- from PGF2α m/z 353 using a quadrupole mass spectrometer 3.8e6 193.1 H - Intensity, cps H H 309.2 164.9 171.2 291.1 208.9 191.1 247.2 111.2 229.2 263.1 124.8 299.2 173.0 255.0 273.2281.2 43.2 59.3 138.8 113.0 137.1 181.2 219.0-44 Da 335.1 353.4 40 100 160 220 280 340 m/z, amu
What information does deuterium labeling at C-2 and C-3 of PGF2 provide us for structure elucidation of PG? Source: Murphy et al. Analytical Biochemistry, 2005
Fragmentation scheme of PGF2α [M-H] - m/z 353 Ions m/z 309, 291, 273 and 193 are indicative of F2-ring
MS/MS fragmentation of PGE2 and PGD2 m/z 351.00 1.9e6 189.0 271.2 Intensity, cps PGE2, Rt 12.07 min 108.9 106.9112.9 135.0 162.9 217.1 67.0 121.0 158.9 174.8 203.9 186.9 191.0 269.3 58.9 95.1 123.0 206.1 235.2 315.0 64.781.1 119.0 137.2157.9170.9 176.9 184.9 228.8 253.3 273.1 296.7 333.3 20 80 140 200 260 320 2.1e6 189.1 Intensity, cps PGD2, Rt 12.4 min 271.1 203.0 59.1 120.9 81.2 94.9106.9 131.6 135.0146.8157.8 173.6186.9191.0 215.1229.0242.8 269.3 289.1 297.2 315.1 20 80 140 200 260 320 m/z, amu
Deuterated PG standards are used for quantitative analysis of PGs in a extract Source: Cao et al. Analytical Biochemistry, 2008
Library search for eicosanoid http://www.lipidmaps.org/
Conclusions Shotgun lipidomics approaches are high throughput and applicable to perform profiling as well as quantitative analysis of various lipids in biological samples. Tandem mass spectrometry analysis of phospholipids in +ve ion mode characterizes phospholipid polar head groups, whereas ve ion mode provide fatty acid chain structural information Identification of phospholipids at a molecular level present a great challenge due to their structural diversity and dynamic metabolism.