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. - PowerPoint PPT Presentation
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, -CEB Microbial Insights, Inc.
Transcript
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,
-CEBMicrobial Insights, Inc.
In-situ Microbial Community Assessment
In the Environment < 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 PCRusing rDNA primers , Separate with denaturing gradient gel electrophoresis (DGGE), sequence for identification and phylogenetic relationship. Great specificity
Lipids: Extract, concentrate, structural analysisQuantitative, Insight into: viable biomass, community composition,Nutritional-physiological status, evidence for metabolic activity
Cathedral from a Brick Predict impact of Cr contamination (from 50-200,000 ppm) on soil microbial community by artificial neural network (ANN) analysis
DNA is “non compressible” ~ perfect code not so influencedBy 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
Signature Lipid Biomarker Analysis
Phospholipid Fatty Acid [PLFA] Biomarker Analysis = Single most quantitative, comprehensive insight into in-situ microbial community
*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)
Feasibility of “Flash” Extraction
ASE vs B&D solvent extraction*
Bacteria = B&D, no distortionFungal Spores = 2 x B&D Bacterial Spores = 3 x B&D Eukaryotic = 3 x polyenoic FA
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
CH2O C
O
CH2(CH2)13CH3
CH2OH
CHO C
O
CH2(CH2)13CH3N
CH3
F+
CH3
SO3
N
CH3
O
N
CH3
CH2O C
O
CH2(CH2)13CH3
CHO C
O
CH2(CH2)13CH3
OCH
CH2O C
O
CH2(CH2)13CH3
CH2
CHO C
O
CH2(CH2)13CH3
Pyridinium Derivative of 1, 2 Dipalmitin
C41H73NO5+
Exact Mass: 659.55
Mol. Wt.: 660.02
C6H7NOExact Mass: 109.05
Mol. Wt.: 109.13
neutral loss
C35H67O4+
Exact Mass: 551.50
Mol. Wt.: 551.90
[M+92]+
[M+92-109]+
M = mass of original Diglyceride
LOD ~100 attomoles/ uL
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
VIABLE NON-VIABLE
O O || ||
H2COC H2COC
| |C O CH C O CH
| |
H2 C O P O CH2CN+ H3
||
|
O
O-
||O
H2 C O H
||O
Polar lipid, ~ PLFA
Neutral lipid, ~DGFA
phospholipase
cell death
Membrane Liability (turnover)
(A) Chromatogram of purified brain and egg yolk derived authentic PG, PE, and PC; (B) Extracted ion chromatogram (EIC) of PG from soil containing 15:0, 16:0, 16:1, 17:0, 17:1, 18:1, 19:1 (see Fig 5); (C) EIC for ions diagnostic of PE from the soil used in B.
A
B
C
PGPE PE
PC
PG
PESeparation on HAISIL reverse phase HL C-18 column, 30 mm x 1mm x 3 μ,95/5 methanol + 0.002% piperidine/water50 μL/min,
post-column modifier 0.02% piperidine in methanol, 10 μL/min.
Parent product ion MS/MS of synthetic PG Q-1 1ppm PG scan m/z 110-990 (M –H) -
Sn1 16:0, Sn2 18:2
Q-3 product ion scan of m/z 747 scanned m/z 110-990 Note 50X > sensitivity
SIM additional 5x > sensitivity ~ 250X
Extract lipids, HPLC/ESI/MS/MS analysis of phospholipids detect specific PLFA as negative ions PLFA 12C Per 13C 16:1 253 269 same as 12C 17:0
Detection of specific per 13C-labeled bacteria added to soils
13C bacteria added
No 13C bacteria added
OHO CH2OH
HO OH
O
CH O
CH2 O
O
O CH2
CH2
CH2 O P
O
O
O-
CH2
CH2
CH2
H2C OH
Archaebacterial Tetraether Lipid
5 ppm
1600 1620 1640 1660 1680 1700 1720 1740m/z0
100
%
1704
1701
1698
16411638
16431695
1664 1680
1706
1707
1713
FW 1640.4
ES+
[M+H]+
[M-2H+Na+K]+
In sim LOQ ~ 50 ppb
[M+H]+
[M+Na]+NOCH3
O
H3OC
O
C9H9NO4Exact Mass: 195.05
Mobile phase: MeOH + 1mM ammonium acetateCone: 40V
ES+
ESI Spectrum of 2, 6-Dimethyl Dipicolinate
LOD ~ 103 spores ~ 0.5 femtomoles/ul
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 DifferenceDaughter ion Fix Vary VaryParent ion Vary Fix VaryNeutral loss Vary Vary FixNeutral gain Vary Vary Fix
Select-ion monitoring Fix Fix Fix
*Collision-induced dissociation (CID) is a reaction region between quadrupoles
Lipid Biomarker Analysis
Tandem Mass Spectrometers
CEB
Ion trap MSn (Tandem in Time)Smaller, Least Expensive, >Sensitive (full scan)
Quadrupole/TOF> Mass Range, > Resolution
MS/CAD/MS (Tandem in Space)1. True Parent Ion Scan to Derivative Ion Scan2. True Neutral Loss Scan 3. Generate Neutral Gain Scan4. More Quantitative 5. > Sensitivity for SIM6. > Dynamic Range
Microbial Insights, Inc.
Problem: Rapid Detection/Identification of Microbes
Propose a Sequential High Pressure/Temperature Extractor Delivers Three Analytes to HPLC/ESI/MS/MS
