Rapid Metabolomic and Lipidomic Analyses Utilizing

Ion Mobility Spectrometry

Erin Shammel BakerXueyun Zheng, Noor A. Aly, Vanessa L. Paurus, Yuxuan Zhou, Berwyck L. J. Poad*, Stephen J. Blanksby*, Richard D. Smith Pacific Northwest National Laboratory*Queensland University of Technology, Brisbane Australia

Understanding health risks

Specific External Environment

General External Environment

Internal Environment

Health Risks

Understanding health risks

Specific External Environment

General External Environment

Internal Environment

Health Risks

1. Small molecules of interest occur from very low to high concentrations (fM-mM) so measurements with high dynamic range and sensitivity are essential

2. Biological changes are best understood when both endogenous metabolites and xenobiotics are analyzed

3. Untargeted measurements covering thousands of small molecules are desired to perform time course studies and analyze large cohorts

Main challenges with small molecule measurements

TestosteroneExact mass = 288.2089188

In the NIST database there are18 different options with exact mass = 288.2089188

4. Many small molecules have the same masses but a different chemical makeup so distinguishing them with MS-based approaches can be difficult or impossible

E

in out

Drift Time

Pulse of 2 ions with same m/z but different shape

Different conformers separate in time with peak heights representing the amount of each

Ion mobility concept

velocity is constant

v = K EK = ion mobility

Drift Cell

Ion mobility conceptLC (minutes) IMS (~60 ms) MS (~100 µs)

IMS MS

Elution Time Drift Time m/z

0 10 20 30 40 50 60

Elution Time (minutes)

Inte

nsity

20 30 40 50 60Drift Time (ms)

20 30 40 50 60Drift Time (ms)

20 30 40 50 60Drift Time (ms)

1100

100

m/z

1100

100

m/z

1100

100

m/z

Isomers difficult to separate with hydrophobic interaction liquid chromatography (HILIC)

D-Glucose-6-phosphate (G6P) α-D-Glucose-1-phosphate (G1P)

HH

D-Fructose-6-phosphate (F6P)

F6PG6P

F6P G6P G1P

G1P

Deprotonated form [M - H]- m/z = 259.02

Different ionization methods

ESI

DNA

Peptides

Drugs

Amino Acids

Sterols, Steroids

Fatty AcidsPAHs

HerbicidesPesticides

APPIAPCI

OH-PAHs

Organic Acids

Sugars

Lipids

PolarityNonpolar Very Polar

Mol

ecul

ar W

eigh

t

10

100

1,000

10,000

Proteins

NucleosidesNucleotides

ESI: Electrospray ionization

Antibiotics

Different ionization methods

ESI

DNA

Peptides

Drugs

Amino Acids

Sterols, Steroids

Fatty AcidsPAHs

HerbicidesPesticides

APPIAPCI

OH-PAHs

Organic Acids

Sugars

Lipids

PolarityNonpolar Very Polar

Mol

ecul

ar W

eigh

t

10

100

1,000

10,000

Proteins

NucleosidesNucleotides

ESI: Electrospray ionization

APCI: Atmospheric pressurechemical ionization

APPI: Atmospheric pressurephotoionization

Antibiotics

BAP: Benzo(a)Pyrene

BKF: Benzo(k)fluoranthene

Radical form [M]·+ m/z = 252.09

Isomeric xenobiotic separations

Pacific Northwest National Laboratory, RichlandAgilent Technologies, Inc., Santa Clara

Vanderbilt University, NashvilleBOKU – Univ. of Natural Resources and Life Sciences, Vienna

IMS collision cross section (CCS) precision

• Compare CCS accuracy across 4 international labs

• Analyze 80 molecules (metabolites, lipids, peptides and proteins) to determine CCS agreement

S. M. Stow, et al., Anal. Chem. 2017, 89, 9048-9055.

%RSD

Lipi

ds

[M-H

]-M

etab

olite

s [M

-H]-

Met

abol

ites

[M+H

]+&

[M+N

a]+

• Ran triplicate injections at all 4 labs

• Analyzed in positive and negative ion mode

• Mean %RSD of 0.24%

m/z

0.00% 0.10% 0.20% 0.30% 0.40% 0.50%C12:0C15:0C16:1C16:0C17:0C18:3C18:2C18:1C18:0C20:4C20:3C20:2C20:1

CreatL-aspartic acidL-glutamic acid

L-histidineL-phenylalanine

Uric AcidL-arginineL-tyrosineL-cystine

Pyr5PMTHF

CreatL-proline

L-threonineH-Cyst

Creat[M+Na]L-histidine

L-phenylalanineL-arginineL-tyrosine

GLC[M+Na]L-cystine

Pyr5PCyst[M+Na]

Pyr5P[M+Na]Cortisol

Cortisol[M+Na]MTHF 95% CIx

_Interlab comparison

Lowest detection at 10 pM for standards in water Linear concentration response

Limit of detection in water using IMS-MS

Lowest detection at 10 nMin human plasma

Lowest detection at 10 pM for standards in water Linear concentration response

Limit of detection in human plasma with IMS-MS

1. Inject Sample

SPE

Cart

ridge

2. Wash Cartridge

SPE

Cart

ridge

3. Reverse FlowSend to MS10 sec analyses

MS

SPE

Cart

ridge

Automated SPEsystem

12 24 36 48 60 72 84 96 108 120Time (s)

wash 6 sample injections/min

1

coun

ts (a

.u.)

2

3

Metabolites extracted from mouse plasma Metabolites extracted from human urine

328.5

328.0

327.5

327.0

18.0 18.5 19.0 19.5 20.0

Isomeric Separation: m/z=327.197

137.9

138.0

138.1

138.2

138.3

13.0 13.4 13.8 14.2

Same nominal mass: m/z=138.054, 138.129

330.0

330.3

330.2

330.1

330.4

19.5 20.0 20.5 21.0 21.5

330.5Isomeric Separation:

m/z=330.228

SPE-IMS-MS analyses of biological samples

~1400 features with S/N > 5 ~1000 features with S/N > 5

X. Zhang, et al., Clin. Mass Spectrom. 2016, 2, 1-10.

2.5

3

3.5

4

4.5

5

5.5

6

2.5 3 3.5 4 4.5 5 5.5

Log1

0 (In

tens

ity)

Log10 (Concentration in pM)

[Imazaquin+H]+

[Hexaconazole+H]+

[Thiabendazole+H]+

[Metribuzin+H]+

[Napropamide+H]+

[Flumeturon+H]+

[Isoxaben+H]+

[Oryzalin+H]+

[Fluroxypyr-1-methylheptyl ester+Na]+

[Resmethrin+Na]+

[Minocycline+H]+

[Fenamidone+H]+

[Fenamiphos+H]+

500 pM

100 nM

Calibration curve for xenobiotics in plasma

X. Zhang, et al., Clin. Mass Spectrom. 2016, 2, 1-10.

Small molecule pipeline

Extraction Instrumental Analysis Data Processing & Analysis

m/z

ID Mass Intensity

---- ---- ----

---- ---- ----

---- ---- ----

---- ---- ----

---- ---- ----

Methanol/Chloroform Extraction

(100 samples/day)

SPE-IMS-MS

(8200 samples/day)

Database Matching &False Discovery Assessment

(? days)

Collision cross section database

100 150 200 250 3000

200

400

600

800

1000

m/z

DTCCSN2 (Å2)

Lipids

Collision cross section database

100 150 200 250 3000

200

400

600

800

1000

m/z

DTCCSN2 (Å2)

>600small molecules

Lipids

X. Zheng, et al., Chem. Sci. 2017, ASAP.

Metabolic pathways

Primary metabolite trend lines

120 160 200 240

200

400

600

800

Amino AcidsNucleotidesSteroids

m/z

DTCCSN2 (Å2)

Protonated form

Primary metabolite trend lines

120 160 200 240

200

400

600

800

Amino AcidsNucleotidesSteroids

m/z

DTCCSN2 (Å2)

120 160 200 240

200

400

600

800

Amino Acids Fatty Acids Lipid Mediators Nucleotides Steroids Sugars

m/z

DTCCSN2 (Å2)

Protonated form Deprotonated form

Website - http://panomics.pnnl.gov/metabolites/

X. Zheng, et al., Chem. Sci. 2017.

Lipidomics

Sterols(Cholesterol)

Polyketides(2ndary Metabolites)

R1

Prenols(vitamins, quinones)

Fatty Acyls(Eicosanoids)

R1

n

Sphingolipids(Ceramides, SM)

Saccharolipids(DGDG, MGDG)

Glycerolipid(MG, DG, TG)

Glycerophospholipids(PC, PE, PG, PS, LP)

Lipid isomers1. Class isomers (PC and PE)

2. cis/trans double bond orientations

3. sn-1/sn-2/sn-3 fatty acyl positions

4. double bond locations

HO

O

HO

O

cis-Δ9 cis-Δ6

sn-2

sn-1 sn-3

PE(18:1/18:1) (cis-Δ9) PE(18:1/18:1) (trans-Δ9)

PE 36:2 < PC 33:2 but has the same elemental composition, C41H78NO8P

PC(14:0/16:0) < PC(16:0/14:0)

<

<

<

Ozonolysis or OzID

+

CriegeeAldehyde

Parent Lipid

Diagnostic IonsΔmass = 16 Da

Steve Blanksby

Ozonolysis on IMS instrument

ESI-OzID-IMS-MS of [PE(18:1/18:1)-H]-

No Ozone Ozone

[M-H]- [M-H]-

Both double bonds trans-Δ9

n-9

n-9, n-9

[M-H]-

[M-H]-

a,ac,a

c,c

ac

ESI-OzID-IMS-MS of PE(18:1/18:1)

[M-H]- [M+H]+

[M-H-110]- [M+H-110]+

cis, cis trans, trans

cis trans

cis, cis trans, trans

cis trans

cis, cis trans, trans

PC 34:1

LC-OzID-IMS-CID-MS in human plasma760.58

PC 34:1

[M-183+H]+

577.52

[M-256+H]+

504.34

599.50496.34[M+H]+

760.58782.57

[M-59+Na]+

723.50

[M-282+H]+

478.33[M+Na]+

PC (16:0/18:1)

NL 59

NL 183

NL 282

NL 2561

Nor

mal

ized

Inte

nsity

LC-OzID-IMS-CID-MS in human plasma760.58

0

600 650 7000.0

0.1N

orm

aliz

ed In

tens

ity

m/z

19.36min

Aldehyde650.44

Criegee666.44

PC 16:0/18:1 (n-9)

600 650 7000.0

0.1

Nor

mal

ized

Inte

nsity

m/z

19.2min

Criegee694.47

Aldehyde678.47

PC 16:0/18:1 (n-7)

[M-183+H]+

577.52

[M-256+H]+

504.34

599.50496.34[M+H]+

760.58782.57

[M-59+Na]+

723.50

[M-282+H]+

478.33[M+Na]+

PC (16:0/18:1)

O

O

OP

O-O

O

N+

O

O

NL 59

NL 183

NL 282

NL 2561

Nor

mal

ized

Inte

nsity

0

PC 34:1760.58

LC-OzID-IMS-CID-MS in human plasma

OzID @ RT 19.2 min OzID @ RT 19.36 min

600 650 7000.0

0.1N

orm

aliz

ed In

tens

ity

m/z

19.36min

Aldehyde650.44

Criegee666.44

PC 16:0/18:1 (n-9)

600 650 7000.0

0.1

Nor

mal

ized

Inte

nsity

m/z

19.2min

Criegee694.47

Aldehyde678.47

PC 16:0/18:1 (n-7)

19.2 19.60

1

Inte

nsity

Retention Time (minute)

760 678 694 650 666

PC 34:1

OzID @ RT 19.2 min OzID @ RT 19.36 min

[M-183+H]+

577.52

[M-256+H]+

504.34

599.50496.34[M+H]+

760.58782.57

[M-59+Na]+

723.50

[M-282+H]+

478.33[M+Na]+

PC (16:0/18:1)

NL 59

NL 183

NL 282

NL 2561

Nor

mal

ized

Inte

nsity

0

LC-OzID-IMS-CID-MS in human plasma

Summary

• Combining multiple separations and methods enables faster and better small molecule identifications as demonstrated with SPE-IMS-MS and LC-OzID-IMS-CID-MS analyses

LC IMS

CID

UVPD

MS

GC

SPE OzID

Acknowledgements• Agilent Technologies• National Institute of Environmental Health

Sciences of the NIH (R01ES022190)• NIH: General Medical Sciences Proteomics

Center at PNNL (2 P41 GM103493-11), National Institute of General Medical Sciences and National Cancer Institute

• PNNL Laboratory Directed Research and Development Program

• Environmental Molecular Sciences Laboratory

Noor Aly

Ion Mobility R&D Group