Metabolomic Profiling of Human Serum: A Case Study for ... · respiratory, vision-related disorders ¾Therapeutic interventions have had limited success, at best, hence the need for

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Metabolomic Profiling of Human Serum:A Case Study for Potential Disease

Biomarker DiscoverySumit Shah, B.Pharm, Ph.D.Metabolomics Applications Scientist,Agilent Technologies Inc.

For Research Use Only. Not for use in diagnostic procedures.

Pre-Term Birth (PTB)PTB is defined as birth before 37 completed weeks of gestation

Incidence rates are on increase over the past three decades (5-7% in developed nations, 9-10% world-wide)

PTB is the leading cause of neonatal mortality and morbidity

Infants born preterm have significant risk of contracting infections(sepsis) and suffering from severe neuro-developmental,respiratory, vision-related disorders

Therapeutic interventions have had limited success, at best,hence the need for better diagnostic tools

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Fetal Umbilical Cord Serum SamplesGroup A are neonates that were delivered at term (Control)

Group B are neonates that were born prematurely (Preterm)

Group C are neonates that were born prematurely and had sepsis(Preterm-Sepsis)

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A

C B


LC-MS based Serum Metabolomics Profiling

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Fetal Umbilical Cord Serum Samples (12 subjects/group)

Liquid-Liquid Extraction (Chloroform/Methanol/Water)

Aqueous ExtractsOrganic Extracts

+ ESI(4μL)

- ESI(12μL)

+ ESI(1μL)

- ESI(10μL)

36 Samples

Group A Group B Group C

36 Samples36 Samples36 Samples

Mixed Mode LCReverse Phase LC

6550 iFunnel Q-TOF 6550 iFunnel Q-TOF

Sample(10μL)

MetaboliteExtraction

(200μL)

Chromatography(1-12μL)

MassSpectrometry

(~50-600nL serum!!)


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TICs for 36 Samples: Organic Extracts, Positive ESI


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LC-MS Data Processing Work-FlowLC-MS Data File (*.d)

MassHunter Qual

MassHunter Output (*.cef)(compound exchange format)

Mass Profiler Professional (MPP)Statistics

ID BrowserPathway Analysis

Batch Analysis DA Reprocessor

RecursiveAnalysis

Molecular FeatureExtractor (MFE)


Data Mining Process

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Monoisotopicmass

(varies in ppm)

Isotope spacing (varies in ppm)

Isotope distribution (varies in %)


Mass Profiler Professional (MPP): Statistics to Pathways and Beyond

Designed for Mass Spectrometry data frommultiple platforms

Import, store, and visualizeAgilent LC/MS TOF, QTOF, and QQQAgilent GC/MS Quad, QQQ, and QTOFAgilent ICP/MSAgilent CE/MSGeneric file format import

Data Filtering and Statistical Tools

ID Browser for compound identification

Pathway Architect for biologicalcontextualization

Sample Class Prediction modeling*

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Compound Alignment: Organic Fractions, +ESI

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11,848 Aligned Features in 36 human serum samples


Data Filtering: Filter by Frequency

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Frequency: Present in at least 9 out of 12 samples in a group

2976 Features after Data Filtering


Fold Change Analysis: [A vs. B], [B vs. C]

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2452 Features exported for Recursive Analysis

A CB


Data Filtering: Filter by Frequency

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Frequency: Present in at least 9 out of 12 samples in each group

2250 Features after Data Filtering on Recursive Data-Set


Statistical Analysis: One-way ANOVA

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1166 Statistically Significant Features

C A BCAB


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Compound Identification with IDBrowser®

Most Extensive Database and MS/MS Library for Metabolomics

METLIN, an accurate mass LC-MS/MS library (Q-TOF based)

Compounds with MS level information – 64092

Lipids with MS level information – 31011

Human metabolites with MS level information – 19714

Compounds with MS/MS spectral information - 8040

MS/MS of mono-isotopic molecular ion

MS/MS in positive and negative ionization mode

Fragmentation performed at three collision energies: 10, 20 and 40

MS/MS spectra are computer and manually reviewed for quality


Summary of Data Analysis

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Sample Fraction Organic Organic Aqueous Aqueous

Ionization +ESI -ESI +ESI -ESI

Aligned Compounds 11848 3092 2891 3144

Frequency Filtered 2976 1299 679 723

Fold-Change > 1.1 2452 973 397 513

Recursive Analysis

Frequency Filtered 2250 948 366 474

Statistics-ANOVA 1166 624 109 187

Database Hits 309/1166 182/624 22/109 63/187


Term vs. Preterm: Most Differentially Expressed Metabolites (25/48) in Aqueous Fractions

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Compound [A Term] [B Preterm] Fold Change Fraction ESI ModeAnatalline 1 4082738 4082737.5 Aqueous Pos5 dihydrotestosterone 1 608300 608300.3 Aqueous Pos4 Heptyloxyphenol 1 462925 462925.1 Aqueous NegLauric acid 1 288129 288129.2 Aqueous Neg2 Ketohexanoic acid 1 286919 286918.5 Aqueous NegJasmonic acid 1 247810 247810.3 Aqueous NegPentadecylic acid 1 197609 197608.7 Aqueous NegGeranyl formate 1 195234 195234.5 Aqueous NegMyclobutanil 1 170861 170861.4 Aqueous NegTriphenylethylene 1 167963 167963.0 Aqueous Negpentan 2 one 1 155456 155455.8 Aqueous NegOrellanine 1 138900 138900.3 Aqueous Pos(S) Dihydroorotate 1 136811 136810.8 Aqueous NegNonanoic acid 1 135823 135823.1 Aqueous Neg5 Dodecenoic acid 1 133063 133063.3 Aqueous Neg4 hydroxynonenal 1 132983 132982.8 Aqueous NegUracil 415889 264127 0.6 Aqueous NegN Butyl 1H pyrazolo[3,4 d]pyrimidin 4 amine 242453 120286 0.5 Aqueous Pos2 Methylerythritol 327046 146653 0.4 Aqueous NegItaconic acid 791270 289118 0.4 Aqueous Negpentadecanal 1144643 321022 0.3 Aqueous PosL Cysteine 766207 163512 0.2 Aqueous NegPalmitic Acid methyl ester 15257650 3145188 0.2 Aqueous Pos4 Hydroxybenzaldehyde 1498240 300809 0.2 Aqueous NegAconitic acid 3122954 494471 0.2 Aqueous Neg


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Compound [A Term] [B Preterm] Fold Change Fraction ESI ModeUlexone B 1 51665908 51665908.0 Lipids PosAntiarone K 1 18950088 18950088.0 Lipids PosPG(O 16:0/0:0) 1 1757852 1757852.1 Lipids PosNervonic acid 1 1550714 1550713.6 Lipids NegPalmityl oleate 1 1112239 1112239.0 Lipids Negsn2 hydroxy archaeol 1 1077817 1077816.8 Lipids NegArachidic Acid 1 1048729 1048728.5 Lipids Neg29:2(5Z,9Z) 1 1033124 1033124.3 Lipids Pos13 methyl 1,2 eicosanediol 1 1016530 1016529.9 Lipids Pos(+) 14 Methyl palmitic acid 1 980877 980877.4 Lipids NegPA(8:0/8:0) 1 963481 963481.4 Lipids PosNonadecyl oleate 1 867908 867907.9 Lipids NegTuberonic acid 1 864406 864406.1 Lipids PosMetagenin 1 822365 822365.3 Lipids Pos20 hydroxy eicosanoic acid 1 795528 795527.9 Lipids PosTridecyl oleate 1 699960 699959.6 Lipids NegN oleoyl threonine 1 695270 695269.7 Lipids PosPI(13:0/14:1(9Z)) 411222 216961 0.5 Lipids Pospristanal 274493 124507 0.5 Lipids Pos4,4 dimethyl 5alpha cholest 7 en 3beta ol 2366936 1033059 0.4 Lipids NegPG(O 16:0/15:1(9Z)) 1715046 701469 0.4 Lipids PosPI(14:1(9Z)/16:1(9Z)) 797498 305035 0.4 Lipids PosPS(12:0/20:3(8Z,11Z,14Z)) 456940 1 456940 Lipids PosPenaresidin A 1359758 1 1359758 Lipids PosHexadecanoic acid, 2 amino , (S) 7693679 1 7693679 Lipids Pos

Term vs. Preterm: Most Differentially Expressed Metabolites (25/289) in Organic Fractions


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Compound [B Preterm] [C Preterm Sepsis] Fold Change Fraction ESI Mode3b,16a Dihydroxyandrostenone sulfate 1380598 6078003 4.4 Aqueous NegCurcumin 1158092 3706188 3.2 Aqueous NegPalmitic Acid methyl ester 3145188 7919997 2.5 Aqueous Pospregnenolone sulfate 205665 483040 2.3 Aqueous Negmethyl (+) 7 isojasmonate 114723 266950 2.3 Aqueous Neg3,4 Dihydroxybenzoic acid 162609 346398 2.1 Aqueous NegDodecanedioic acid 743387 1388629 1.9 Aqueous Neg3 Hydroxydodecanedioic acid 5592027 10153707 1.8 Aqueous Neg3 Hydroxytetradecanedioic acid 17796788 32004686 1.8 Aqueous NegKhellol glucoside 319747 503233 1.6 Aqueous PosUric acid 3551611 5334889 1.5 Aqueous PosPhenkapton 623917 927324 1.5 Aqueous Pos1alpha,5alpha Dimercaptoandrostane 3alpha,17beta diol 552939 816082 1.5 Aqueous Neg2 Methyl 3 hydroxy 5 formylpyridine 4 carboxylate 281082 156481 0.6 Aqueous Neg2,4 Dichlorobenzoate 1826074 1005932 0.6 Aqueous NegL Capreomycidine 511794 263227 0.5 Aqueous Pos4 Methylumbelliferyl ? D glucuronide 440712 223826 0.5 Aqueous NegCreatine 6572541 3229358 0.5 Aqueous Pos3 Hydroxyhippuric acid 298386 145353 0.5 Aqueous NegAnatalline 4082738 1964418 0.5 Aqueous Postrans Cinnamoyl beta D glucoside 764550 325086 0.4 Aqueous NegD Mannitol 18114186 5500759 0.3 Aqueous PosD Mannitol 8021985 2167221 0.3 Aqueous NegCitric acid 438111 115561 0.3 Aqueous NegCitric acid 661972 150045 0.2 Aqueous Pos(R) 2 Hydroxypropyl CoM 2313157 333988 0.1 Aqueous NegSalicylamide 135725 1 135725 Aqueous Neg2 Methylerythritol 146653 1 146653 Aqueous Neg

Preterm vs. Preterm-Sepsis: 26 Differentially Expressed Metabolites in Aqueous Fractions


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Compound [B Preterm] [C Preterm Sepsis] Fold Change Fraction ESI ModePenaresidin A 1 721182 721181.5 Lipids PosCytochalasin B 128475 474831 3.7 Lipids PosMID42085:(6RS) 6,19 epidioxy 24,24 difluoro 25 hydroxy 6,19dihydrovitamin D3 311104 1143727 3.7 Lipids Pos4,2',4',6' Tetrahydroxy 3,5 diprenyldihydrochalcone 325273 962383 3.0 Lipids Pos7E,9Z,11 Dodecatrienyl acetate 1063085 2762442 2.6 Lipids Neg16:4(4Z,7Z,10Z,13Z) 265404 597244 2.3 Lipids NegPE(21:0/20:2(11Z,14Z)) 4493165 9718519 2.2 Lipids PosPS(22:2(13Z,16Z)/22:2(13Z,16Z)) 875262 1826514 2.1 Lipids NegTocopherol 309579 609879 2.0 Lipids PosPS(22:4(7Z,10Z,13Z,16Z)/22:1(11Z)) 3506648 6877293 2.0 Lipids Neg12 oxo LTB4 667574 1304543 2.0 Lipids NegCer(d18:0/24:0) 2174600 4249472 2.0 Lipids NegPE(21:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 6842784 13370377 2.0 Lipids Poslithocholic acid sulfate 1174682 2277477 1.9 Lipids PosPA(20:1(11Z)/0:0) 1414219 2732446 1.9 Lipids PosPI(16:1(9Z)/22:2(13Z,16Z)) 244072 465958 1.9 Lipids NegCer(d18:0/24:0) 829529 1576760 1.9 Lipids PosAnandamide (20:2, n 6) 8307423 1159227 0.1 Lipids PosDocosatetraenoyl Ethanolamide 2543583 308339 0.1 Lipids Pos7 Heptadecynoic acid 121820 1 121820 Lipids Pos2 hydroxy heptadecanoic acid 185291 1 185291 Lipids Pos15 hydroxy heneicosanoic acid 243210 1 243210 Lipids Pos2 Methyl tridecanoic acid 251484 1 251484 Lipids NegN arachidonoyl leucine 277669 1 277669 Lipids Pos32:6(14Z,17Z,20Z,23Z,26Z,29Z) 282960 1 282960 Lipids Pos

Preterm vs. Preterm-sepsis: Most Differentially Expressed Metabolites (25/169) in Organic Fractions


Citric Acid (or Isocitric Acid)

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Citric Acid

A CB


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Citric AcidGroup A (12 samples) Group C (12 samples)Group B (12 samples)


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TCA Cycle


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Pathway for Metabolism of Amino acids and Derivatives


ConclusionsMetabolomic profiling revealed significant differences betweenthe neonates that were born term vs. prematurely, as well asbetween prematurely born neonates that did or did not havesepsis

337 metabolites were found to be significantly different (p-value< 0.05, Fold-change > 1.5) between the Term and Preterm bornneonates

195 metabolites were found to be significantly different (p-value< 0.05, Fold-change > 1.5) between the prematurely bornneonates that did or did not have sepsis

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Moving Forward……

Repeat the work in a separate sample-set to confirm thepanel of significant features/metabolites

Perform MS/MS analysis to confirm the identity of the panelof annotated metabolites

Develop an LC-MRM/MS quantitation assay for highthroughput screening of the metabolite panel in a largercohort of samples

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Acknowledgements

Ian A. Blair

Michal Elovitz

Jasbir S. Arora

Jim Lau

Steve Fischer

Susanne Moyer

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Grant #21FY08-539 (Michal Elovitz)


Documents

Metabolomic Profiling of Human Serum: A Case Study for ... · respiratory, vision-related disorders ¾Therapeutic interventions have had limited success, at best, hence the need for