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 orDiseased group
Control group
Urine, plasma, tissueetc.
Sample preparation Internal standard spiking for quantitative analysis Extraction (liquid-liquid, ppt with or without hydrolysis)
Sample analysisLC-MS and LC-MS/MS analysis• Q1 scan using HPLC or UPLC/-TOFMS• +ve/-ve ion mode- accurate mass measurement• MS/MS experiments using a hybrid instrument Q-Trap
Data analysisMultivariat analysis e.g. PCA
Data export
Marker identification
Lipidomics can perhaps best be defined as a comprehensive analysis of lipids on the systems-level scale together with their interacting factors
Genome Transcriptome Proteome Metabolome
What might be happening in a cellSnapshot of the entirephysiology
Lipidomics
Metabolomics in the context of other omics
Lipids are important- as a membrane bilayer- provides hydrophobic environment for protein function- reservoir of energy- signaling molecules
Outlines
• 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
CH2OHOH
NHR
OCeramide
NH2
N(CH3)3
OHOH
NH2OH
O
HO
OH
HO
OH
OH
Phosphatidylethanolamine (PE)Y=
Phosphatidylcholine (PC)Y=
Phosphatidylglycerol (PG)Y=
Phosphatidylserine (PS)Y=
Phosphatidylinositol (PI)Y=
O
O
O
OOP
OHO
OHO
OP
OOH
OO
O
O
O
Structures of main phospholipids
Sn-1
Sn-2
Cardiolipin (diphosphatidylglycerol)
Sn-3
Choline Phosphocholine Cytidine diphosphocholine
1,2-diacylglycerol
Phosphatidylcholine
Activation of phospholipase A2
Lysophosphatidylcholine (LPC)
+ Free fatty acid
Pro-inflammatoryproperty
Component of oxidized LPL
PhosphatidylethanolamineHydrolysis by phospholipase A2
Lysophosphatidylethanolamine(LPE)
How phospholipids are synthesized?
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 oC untill 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 PELPC PAPE PILPE PISM PGPS PIPs
PC = phosphatidylcholinePA = phosphatic acidPE = phosphatidylethanolaminePS = phosphatidylserinePG = phosphatidylglycerolPI = phosphatidylinositolPIP = PI monophosphateSM = sphingomyelinLPE = lysoPE
Increasing metabolite coverage using +ve and –ve ion mode
Source: Nordstrom et al. Analytical Chemistry, 2007
No arachidonicAcid in +ve ion mode
Representative Q1 scans of a methanolic extract of human blood serum
Application of shotgun lipidomics: intra-source separation of lipids
Source: Gross and Han, methods in Enzymology, 2007
400 475 550 625 700 775 850 900m/z0
100
%
518.345496.361
494.352
758.599
542.347544.367
546.380
566.351568.366
590.351756.584591.359 703.605
786.632
806.604810.628
811.636812.644
835.632
869
Total scan of metabolites (Q1 SCAN + ion mode) for a plasma sample obtained from lean mouse [A]; ob/ob mouse
400 475 550 625 700 775 850 m/z0
100
%
518.318
496.335
494.326
760.570758.553544.339
546.352566.322
568.337590.321
732.558602.288
782.552
806.556810.592
811.599812.608
835.594
[A]
[B] 1.52e3
Total metabolomics
20.3
400 475 550 625 700 775 850 900m/z0
100
%
480.425
478.419
504.432
508.464
558.466885.817820.780794.751588.418 646.397 732.383674.430 830.818 886.779
400 475 550 625 700 775 850 900m/z0
100
%
52.5480.415
504.431506.445540.459508.463
564.474588.456 794.760816.775
Total scan of metabolites (Q1 SCAN -ve ion mode) for a plasma sample obtained from lean mouse [A]; ob/ob mouse
m/z 184
Phosphatidylethanolamine
Tandem mass spectrometry (MS/MS) of phospholipids
185 Phosphatidylserine
P
O-O
O
NH2O
H OR1
O
OR2
O 141
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
400 460 520 580 640 700 760 820 880
2.7e6
Intensity, cps
759.1
783.0787.1 807.1761.1
811.1762.1496.9
788.2525.0 835.1704.1 781.1 814.1544.8 733.0 836.1568.8 795.4 805.0767.9702.0546.9494.8 690.0
+Prec (184.00): Exp 1, 2.764 to 30.604 min from Sample 11 (Lean 53 NK) of ZhangSET1.wiff (Turbo Spray), Centroided
400 460 520 580 640 700 760 820 880
3.1e6
Intensity, cps
807.1761.1783.0
759.1
787.0496.9
808.1811.1
525.0522.9 704.1520.8 789.1 835.1781.1757.0 814.2544.7 733.0 805.0568.8 791.3 837.1546.9 702.0494.8 769.3 817.2797.0747.0
+Prec (184.00): Exp 1, 2.764 to 30.603 min from Sample 16 (Ob 27 NK) of ZhangSET1.wiff (Turbo Spray), Centroided
400 460 520 580 640 700 760 820 880
3.1e6
Intensity, cps
807.1761.1783.0
759.1
787.0496.9
808.1811.1
525.0522.9 704.1520.8 789.1 835.1781.1757.0 814.2544.7 733.0 805.0568.8 791.3 837.1546.9 702.0494.8 769.3 817.2797.0747.0
+Prec (184.00): Exp 1, 2.764 to 30.603 min from Sample 16 (Ob 27 NK) of ZhangSET1.wiff (Turbo Spray), Centroided
Intensity, cps
400 460 520 580 640 700 760 820 880
6239769.0
744.9
778.8764.9
745.8478.3482.5 750.8 779.8740.9716.9502.6
526.5454.5483.5 792.8
751.8636.8 729.1 762.8703.0 738.9548.5 661.7690.8 797.0528.7430.1 476.5 606.6508.4 810.8600.5583.7452.4 619.5 732.2 892.5
+NL (141.00): Exp 2, 2.278 to 33.485 min from Sample 11 (Lean 53 NK) of ZhangSET1.wiff (Turbo Spray), Centroided
400 460 520 580 640 700 760 820 880 900
3400
Intensity, cps
769.0
764.9769.9482.5
454.5 740.9480.5 502.5 716.8478.5483.5 526.5 792.9778.9762.7
794.9738.8659.3528.6430.0 600.4 752.8524.4 687.4636.8548.3 795.9452.4 715.9 786.7 836.9426.1 898.8663.6581.7 812.9 857.9
+NL (141.00): Exp 2, 2.278 to 33.485 min from Sample 15 (Ob 26 NK) of ZhangSET1.wiff (Turbo Spray), Centroided
[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 = ?
[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
MSMS fragmentation of m/z 496 obtained from plasma of ob/ob mousesupplemented with kudzu
TOF MSMS 494.35ES+
75 150 225 300 375 450 525 m/z0
100
%
184.080
104.113
86.104 478.348
HOP
HOO
Om/z 125
125 220 280 340 400 460 520m/z0
100
%
478.348
313.285
258.118
239.247 311.266283.283
419.277
417.264 476.331496.357
-59
-18
MS/MS of m/z 480 [M-15]- from a ob/ob no kudzu supplemented plasma sample TOF MSMS 480.40ES-
220 280 340 400 460m/z0
100
%
255.267
224.102242.116
256.275
+
m/z 480, [M-15]-
C16:0
O
O
OH
O POH
OO
N m/z 524LysoPC
O
O
O
O POH
OO
N
O
m/z 524
O
O POH
OO
N
O
O m/z 524
Platelet activating factor
LysoPC
sn-1
sn-2
Several isomeric compounds exits and unambiguous identification is a challenge
Source: Hsu et al. J. Am Soc. Mass Spectrom, 1998
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 isomersof lithiated phospholipids
A 2D ESI mass spectrometric finger print for TG molecules
Source: Han and Gross, 2004
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
MS/MS analysis of eicosanoids
Structural representation PG based on ring features
R = aliphatic chain
Intensity, cps
m/z, amu40 100 160 220 280 340
3.8e6193.1
309.2164.9
171.2 291.1208.9
247.2191.1111.2 353.4263.1
255.0173.0229.2
273.2137.1 181.2
281.2219.043.2 113.0
124.8 299.2
335.159.3 138.8
ESI-MS/MS of the [M-H]- from PGF2α
m/z 353 using a quadrupole mass spectrometer
-44 Da
O-
O
OH
HO
HO
Source: Murphy et al. Analytical Biochemistry, 2005
What information does deuterium labeling at C-2 and C-3 ofPGF2 provide us for structure elucidation of PG?
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
m/z, amu
20 80 140 200 260 320
1.9e6
Intensity, cps
189.0
271.2
108.9112.9106.9 135.0 162.9 217.1174.8 203.9121.0 158.967.0 191.0186.9 269.395.1 333.3206.1 315.0123.0 235.2170.9 228.8137.2 253.3119.0 184.981.1 157.958.9 176.964.7 296.7273.1
20 80 140 200 260 320
2.1e6
Intensity, cps
189.1
271.1
203.0120.959.1 157.8 186.9 242.8135.0106.9 215.1173.681.2 94.9 191.0 289.1269.3229.0146.8 297.2 315.1131.6
PGD2, Rt 12.4 min
PGE2, Rt 12.07 min
Deuterated PG standards are used for quantitativeanalysis 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.