Expanded Utility of Signature Lipid Biomarker Analysis for

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Expanded Utility of Signature Lipid Biomarker
Analysis for Microbial Community Composition and
Nutritional/Physiological Status with
HPLC/ESI/MS/MS Analysis of Intact Lipid Components
David C. White, Cory Lytle, Sarah J.
Macnaughton, John R. Stephen, Aaron Peacock,
Carol A. Smith, Ying Dong Gan, Yun-Juan Chang,
Yevette M. Piceno Center for Environmental
Biotechnology, University of Tennessee,
Knoxville, TN, Environmental Sciences Division,
Oak Ridge National Laboratory, Oak Ridge, TN,
Microbial Insights, Inc., Rockford, TN,

Microbial Insights, Inc.
-CEB
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In-situ Microbial Community Assessment
In the Environment lt 1.0 to 0.1 of the in-situ
microbial community is detected using Isolation
and Classical Plate Count Many non-culturable
organisms can be infectious (VNCB), isolation can
take days, lose insight into community
interactions physiology Two Complimentary
Biomarker Methods DNA Recover from
surface, Amplify with PCR using rDNA primers ,
Separate with denaturing gradient gel
electrophoresis (DGGE), sequence for
identification and phylogenetic relationship.
Great specificity Lipids Extract, concentrate,
structural analysis Quantitative, Insight into
viable biomass, community composition, Nutritional
-physiological status, evidence for metabolic
activity
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Signature Lipid Biomarker Analysis
Cathedral from a Brick Predict impact of Cr
contamination (from 50-200,000 ppm) on soil
microbial community by artificial neural network
(ANN) analysis PLFA (phospholipid fatty acid)
excellent x 102-103 ppm Cr with (PLFA). DNA
is non compressible perfect code not so
influenced By microniche conditions as cell
membranes PLFA is compressible as contains
physiological status input Contains holistic
information responds to perturbations
Predict it is a Cathedral or a Prison DNA a
perfect brick PLFA a non-linear mixture of
bricks and a window

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Signature Lipid Biomarker Analysis

• Phospholipid Fatty Acid PLFA Biomarker
  Analysis Single most quantitative,
  comprehensive insight into in-situ microbial
  community
• Why not Universally utilized?
• Requires 8 hr extraction with ultrapure solvents
  emulsions.
• Ultra clean glassware incinerated 450oC.
• Fractionation of Polar Lipids
• Derivatization transesterification
• 5. GC/MS analysis picomole detection 104
  cells LOD
• 6. Arcane Interpretation Scattered Literature
• 7. 3-4 Days and 250

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Signature Lipid Biomarker Analysis
Expand the Lipid Biomarker Analysis
1. Increase speed and recovery of extraction
Flash 2. Include new lipids responsive to
physiological status HPLC (not need
derivatization increase molecular
size) Respiratory quinone redox terminal
electron acceptor Diglyceride cell
lysis Archea methanogens Lipid ornithine
bioavailable phosphate Lysyl-phosphatidyl
glycerol low pH Poly beta-hydroxy alkanoate
unbalanced growth 3. Increased Sensitivity
and Specificity ESI/MS/MS

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Lyophilized Soil Fractions, Pipe Biofilm
1. Neutral Lipids
SFECO2
UQ isoprenologues
ESE Chloroform.methanol
Derivatize N-methyl pyridyl Diglycerides
Sterols Ergostrerol Cholesterol
2. Polar Lipids
Transesterify PLFA
Intact Lipids
Phospholipids PG, PE, PC, Cl, sn1
sn2 FA Amino Acid PG Ornithine lipid Archea ether
lipids Plamalogens
3. In-situ Derivatize in SFECO2
CG/MS
PHA Thansesterify Derivatize N-methyl
pyridyl
2,6 DPA (Spores)
LPS-Amide OH FA
HPLC/ESI/MS/MS
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Lipid Biomarker Analysis
Sequential High Pressure/Temperature Extraction
( 1 Hour) Supercritical CO2 Methanol enhancer
Neutral Lipids, (Sterols, Diglycerides,
Ubiquinones) Lyses Cells
Facilitates DNA Recovery (for off-line
analysis 2. Polar solvent Extraction
Phospholipids CID detect negative ions
Plasmalogens Archeal Ethers 3). In-situ
Derivatize Extract Supercritical CO2 Methanol
enhancer 2,6 Dipicolinic acid
Bacterial Spores Amide-Linked Hydroxy
Fatty acids Gram-negative LPS Three
Fractions for HPLC/ESI/MS/MS Analysis
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Feasibility of Flash Extraction
ASE vs BD solvent extraction Bacteria BD,
no distortion Fungal Spores 2 x BD Bacterial
Spores 3 x BD Eukaryotic 3 x polyenoic
FA 2 cycles 80oC, 1200 psi, 20 min vs BD
8 -14 Hours
Macnaughton, S. J., T. L. Jenkins, M. H. Wimpee,
M. R. Cormier, and D. C. White. 1997. Rapid
extraction of lipid biomarkers from pure culture
and environmental samples using pressurized
accelerated hot solvent extraction. J.
Microbial Methods 31 19-27(1997)
CEB
Microbial Insights, Inc.
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ESI (cone voltage)
Q-1
CID
Q-3
FRAGMENTATION with ESI/MS/MS
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Respiratory Ubiquinone (UQ)
Gram-negative Bacteria with Oxygen as terminal
acceptor LOQ 225 femtomole/uL, LOD 75
femtomole/uL 100 E. coli
Isocratic 95.5/4.5 methanol/aqueous 1 mM
ammonium acetate
Q7
Q10
Q6
197 m/z
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Pyridinium Derivative of 1, 2 Dipalmitin
M92-109
M mass of original Diglyceride LOD 100
attomoles/ uL
M92
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LIPID Biomarker Analysis
1. Intact Membranes essential for Earth-based
life 2. Membranes contain Phospholipids 3.
Phospholipids have a rapid turnover from
endogenous phospholipases . 4. Sufficiently
complex to provide biomarkers for viable
biomass, community composition,
nutritional/physiological status 5. Analysis
with extraction provides concentration
purification 6. Structure identifiable by
Electrospray Ionization Mass Spectrometry at
attomoles/uL (near single bacterial cell) 7.
Surface localization, high concentration ideal
for organic SIMS mapping localization
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Membrane Liability (turnover)
VIABLE
NON-VIABLE
O
O


H2COC
H2COC
O
O
phospholipase




cell death
C O CH
C O CH

O


H2 C O H
H2 C O P O CH2CN H3

Neutral lipid, DGFA
O-
Polar lipid, PLFA
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PE
PE
PG
A
PC
PG
B
Separation on HAISIL reverse phase HL C-18
column, 30 mm x 1mm x 3 µ, 95/5 methanol 0.002
piperidine/water 50 µL/min, post-column modifier
0.02 piperidine in methanol, 10 µL/min.
PE
C
(A) Chromatogram of purified brain and egg yolk
derived authentic PG, PE, and PC (B) Extracted
ion chromatogram (EIC) of PG from soil containing
150, 160, 161, 170, 171, 181, 191 (see Fig
5) (C) EIC for ions diagnostic of PE from the
soil used in B.
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Parent product ion MS/MS of synthetic PG
Q-1 1ppm PG scan m/z 110-990
(M H) -
Sn1 160, Sn2 182
Q-3 product ion scan of m/z 747 scanned m/z
110-990 Note 50X gt sensitivity
SIM additional 5x gt sensitivity 250X
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Detection of specific per 13C-labeled bacteria
added to soils
Extract lipids, HPLC/ESI/MS/MS analysis of
phospholipids detect specific PLFA as
negative ions PLFA 12C Per 13C
161 253 269 same as
12C 170 160 255 271 Unusual
12C 170 (269) 2 13C ? cy170 267
284 12C 180 (283) 13C 181 281
299 12C 206 , 12C 190 with 2 13C
? 191 295 314 12C 215 (315),
12C 216 (313)
?
13C bacteria added
?
No 13C bacteria added
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Archaebacterial Tetraether Lipid
FW 1640.4
In sim LOQ 50 ppb
ES
M-2HNaK
MH
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ESI Spectrum of 2, 6-Dimethyl Dipicolinate
LOD 103 spores 0.5 femtomoles/ul
MH
ES
Mobile phase MeOH 1mM ammonium acetate Cone
40V
MNa
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Lipid Biomarker Analysis
Expanded Lipid Analysis Greatly Increase
Specificity Electrospray Ionization ( Cone
voltage between skimmer and
inlet ) In-Source Collision-induced dissociation
(CID) Tandem Mass Spectrometry Scan
Q-1 CID Q-3
Difference Daughter ion Fix Vary
Vary Parent ion Vary
Fix Vary Neutral loss
Vary Vary Fix Neutral
gain Vary Vary
Fix Select-ion monitoring Fix
Fix Fix Collision-induced
dissociation (CID) is a reaction region between
quadrupoles

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Tandem Mass Spectrometers
Ion trap MSn (Tandem in Time) Smaller, Least
Expensive, gtSensitive (full scan)
Quadrupole/TOF gt Mass Range, gt Resolution
MS/CAD/MS (Tandem in Space) 1. True Parent Ion
Scan to Derivative Ion Scan 2. True Neutral
Loss Scan 3. Generate Neutral Gain Scan 4.
More Quantitative 5. gt Sensitivity for SIM 6.
gt Dynamic Range
Microbial Insights, Inc.
CEB
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Problem Rapid Detection/Identification of
Microbes
Propose a Sequential High Pressure/Temperature
Extractor Delivers Three Analytes to
HPLC/ESI/MS/MS