Rapid sample analyses for environmental toxicants Erin Shammel Baker - - PowerPoint PPT Presentation

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Rapid sample analyses for environmental toxicants Erin Shammel Baker - - PowerPoint PPT Presentation

Oct 10, 2022 169 likes •487 views

Rapid sample analyses for environmental toxicants Erin Shammel Baker Xueyun Zheng, Noor A. Aly, Francesca B. Smith, Kristin E. Burnum-Johnson, Samuel H. Payne, Matthew E. Monroe, Richard D. Smith Pacific Northwest National Laboratory Understanding

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SLIDE 1

Rapid sample analyses for environmental toxicants

Erin Shammel Baker Xueyun Zheng, Noor A. Aly, Francesca

  • B. Smith, Kristin E. Burnum-Johnson, Samuel
  • H. Payne, Matthew E. Monroe, Richard D. Smith

Pacific Northwest National Laboratory

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SLIDE 2

Understanding health risks

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SLIDE 3

Understanding health risks

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SLIDE 4

Understanding health risks

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SLIDE 5

Understanding health risks

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SLIDE 6

Understanding health risks

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SLIDE 7

Multi-omic analyses

  • Human genome project

illustrated that >90% of diseases are not due solely to genetics

MS-based Measurements

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SLIDE 8

Main challenges with small molecule measurements

  • 1. Small molecules occur from very low to high concentrations (fM-mM) so high

dynamic range and sensitivity measurements 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 for

time course studies and large cohort analyses

  • 4. Many small molecules have the same masses but

a different chemical makeup so distinguishing them with MS-based approaches can be difficult

  • r impossible

Testosterone In the NIST database there are Exact mass = 288.2089188 18 different

  • ptions with exact

mass = 288.2089188

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SLIDE 9

Ion mobility concept

E

in

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

Drift Cell

velocity is constant v = K E K = ion mobility

  • ut

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

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SLIDE 10

Ion-neutral collision cross section (CCS)

1. Value related to the size and shape of an ion

  • 2. Corresponds to the area

that collides with the drift gas 3. Robust physicochemical property 4. Can easily be compared between labs 5. Varies depending on drift gas

Slide courtesy of Professor Kevin Pagel and Waters

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SLIDE 11

1100 100 m/z

Ion mobility concept

MS (~100 µs) LC (minutes) IMS (~60 ms)

IMS Elution Time Drift Time MS m/z

20 30 40 50 60 100 m/z 1100 1100 Drift Time (ms) 20 30 40 50 60 100 m/z

10 20 30 40 50 60 Intensity

20 30 40 50 60 Drift Time (ms) Drift Time (ms)

Elution Time (minutes)

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SLIDE 12

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

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

H H

D-Fructose-6-phosphate (F6P) F6P G6P G1P

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

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SLIDE 13

Isomeric xenobiotic separations

Collaboration with Keri Hornbuckle’s Group

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SLIDE 14

Isomeric xenobiotic separations

Collaboration with Keri Hornbuckle’s Group

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SLIDE 15

Pacific Northwest National Laboratory, Richland Agilent Technologies, Inc., Santa Clara Vanderbilt University, Nashville BOKU – 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.
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SLIDE 16

%RSD

Lipids [M-H]- Metabolites [M-H]- Metabolites [M+H]+ & [M+Na]+

  • 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% C12:0 C15:0 C16:1 C16:0 C17:0 C18:3 C18:2 C18:1 C18:0 C20:4 C20:3 C20:2 C20:1 Creat L-aspartic acid L-glutamic acid L-histidine L-phenylalanine Uric Acid L-arginine L-tyrosine L-cystine Pyr5P MTHF Creat L-proline L-threonine H-Cyst Creat[M+Na] L-histidine L-phenylalanine L-arginine L-tyrosine GLC[M+Na] L-cystine Pyr5P Cyst[M+Na] Pyr5P[M+Na] Cortisol Cortisol[M+Na] MTHF

95% CI x _

Interlab precision comparison

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SLIDE 17

Interlab mixture reproducibility

Sample 1 Sample 2 Sample 3

Collaboration with Ivan Rusyn’s Group

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SLIDE 18
  • 1. Inject Sample

SPE Cartridge

  • 2. Wash Cartridge

SPE Cartridge

  • 3. Reverse Flow

Send to MS 10 sec analyses MS SPE Cartridge

Automated SPE system

12 24 36 48 60 72 84 96 108 120

Time (s) wash 6 sample injections/min

1

counts (a.u.)

2 3

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SLIDE 19

Polar molecules extracted from mouse plasma Polar molecules 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 21.5 330.5

Isomeric 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.
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SLIDE 20

2.5 3 3.5 4 4.5 5 5.5 6 2.5 3 3.5 4 4.5 5 5.5 Log10 (Intensity) 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.
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SLIDE 21

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)

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SLIDE 22

Collision cross section database

100 150 200 250 300 200 400 600 800 1000

m/z

DTCCSN2 (Å 2)

Lipids

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SLIDE 23

Collision cross section database

100 150 200 250 300 200 400 600 800 1000

m/z

DTCCSN2 (Å 2)

>600 small molecules Lipids

  • X. Zheng, et al., Chem. Sci. 2017, ASAP.
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SLIDE 24

Metabolic pathways

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SLIDE 25

Primary metabolite trend lines

120 160 200 240 200 400 600 800

Amino Acids Nucleotides Steroids

m/z

DTCCSN2 (Å 2)

Protonated form

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SLIDE 26

Primary metabolite trend lines

120 160 200 240 200 400 600 800

Amino Acids Nucleotides Steroids

m/z

DTCCSN2 (Å 2)

120 160 200 240 200 400 600 800

Amino Acids Fatty Acids Lipid Mediators Nucleotides Steroids Sugars

m/z

DTCCSN2 (Å 2)

Protonated form Deprotonated form

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SLIDE 27

Xenobiotic trend lines

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SLIDE 28

L-Tyrosine (amino acid) Exact mass: 181.0738896 Glufosinate (herbicide) Exact mass: 181.0503922

16 17 18 19 20 1

Normalized Intensity Arrival time (ms) [Tyrosine - H]- [Glufosinate - H]-

Deprotonated form 134.7 Å2 147.5 Å2

Primary metabolite versus xenobiotic

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SLIDE 29

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

  • X. Zheng, et al., Chem. Sci. 2017.
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SLIDE 30

Summary

  • Ion mobility spectrometry enables rapid structural analyses
  • Combining multiple separations and methods enables faster and better small molecule

identifications (i.e. SPE-IMS-MS)

Phenomics

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SLIDE 31

Acknowledgements

  • Agilent Technologies
  • NIEHS R01ES022190
  • NIEHS Superfund Research Program P42

ES027704

  • NIH General Medical Sciences P41

GM103493-11

  • PNNL Laboratory Directed Research and

Development Program

  • Environmental Molecular Sciences

Laboratory

Noor Aly

Ion Mobility R&D Group