Transcript Document

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
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 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
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
Signature Lipid Biomarker Analysis
Phospholipid Fatty Acid [PLFA] Biomarker Analysis =
Single most quantitative, comprehensive insight into insitu microbial community
Why not Universally utilized?
1. Requires 8 hr extraction with ultrapure solvents [emulsions].
2. Ultra clean glassware [incinerated 450oC].
3. Fractionation of Polar Lipids
4. Derivatization [transesterification]
5. GC/MS analysis ~ picomole detection ~ 104 cells LOD
6. Arcane Interpretation [Scattered Literature]
7. 3-4 Days and ~ $250
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
Lyophilized Soil Fractions, Pipe Biofilm
1. Neutral Lipids
SFECO2
UQ isoprenologues
ESE Chloroform.methanol
2. Polar Lipids
Transesterify
Intact Lipids
Derivatize –N-methyl pyridyl
Diglycerides
Sterols
Ergostrerol
Cholesterol
PLFA
CG/MS
Phospholipids
PG, PE, PC, Cl,
& sn1 sn2 FA
Amino Acid PG
Ornithine lipid
Archea ether lipids
Plamalogens
3. In-situ Derivatize in SFECO2
PHA
Thansesterify &
Derivatize
N-methyl pyridyl
2,6 DPA (Spores)
LPS-Amide OH FA
HPLC/ESI/MS/MS
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
Feasibility of “Flash” Extraction
ASE vs B&D solvent extraction*
Bacteria = B&D, no distortion
Fungal Spores = 2 x B&D
Bacterial Spores = 3 x B&D
Eukaryotic = 3 x polyenoic FA
[2 cycles 80oC, 1200 psi, 20 min]
vs B&D = 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)
Microbial
Insights, Inc.
CEB
ESI
(cone voltage)
Q-1
CID
Q-3
FRAGMENTATION with ESI/MS/MS
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
Q6
Q10
O
H3 O C
CH3
H3 O C
O
197 m/z
H
]n
Pyridinium Derivative of 1, 2 Dipalmitin
SO3
O
CH2O C
CH2(CH2)13CH3
O
C
CH2(CH2)13CH3
CHO
+
F
N
CH3
CH3
[M+92-109]+
CH2OH
O
CH2O C
CH2(CH2)13CH3
O
C
CH2(CH2)13CH3
CHO
C6H7NO
Exact Mass: 109.05
Mol. Wt.: 109.13
neutral loss
C41H73NO5+
Exact Mass: 659.55
Mol. Wt.: 660.02
OCH
N
CH3
O
N
CH3
M = mass of original
Diglyceride
O
CH2O C
CH2(CH2)13CH3
O
C
CH2(CH2)13CH3
CHO
CH2
C35H67O4+
Exact Mass: 551.50
Mol. Wt.: 551.90
LOD ~100 attomoles/ uL
[M+92]+
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
Membrane Liability (turnover)
VIABLE
NON-VIABLE
O
O
||
||
H2COC
O H2COC
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
OPolar lipid, ~ PLFA
PG
PG
C
PE
PE
PC
A
B
PE
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.
(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.
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
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
16:1 253
269
same as 12C 17:0
16:0 255
cy17:0 267
18:1 281
19:1 295
271 Unusual 12C 17:0 (269) + 2 13C
284 12C 18:0 (283) + 13C
299
314
13C
12C
20:6 , 12C 19:0 with 2 13C
12C 21:5 (315), 12C 21:6 (313)
bacteria added

No 13C bacteria added



Archaebacterial Tetraether Lipid
HO
HO
CH 2OH
O
O
OH
CH2
CH
O
O
O
CH 2 O
CH 2
CH 2
H2C
OH
CH 2
O
CH 2
O-
CH 2 O P
O
FW 1640.4
In sim LOQ ~ 50 ppb
5 ppm
ES+
1704
100
[M-2H+Na+K]+
1701
1698
[M+H]+
%
1707
16381641
1643
0
1600
1620
1640
1706
1664
1660
1680
1680
1695
1713
1700
m/z
1720
1740
ESI Spectrum of 2, 6-Dimethyl Dipicolinate
LOD ~ 103 spores ~ 0.5 femtomoles/ul
[M+H]+
ES+
Mobile phase: MeOH + 1mM ammonium acetate
Cone: 40V
H3OC
OCH3
N
O
O
C9H9NO4
Exact Mass: 195.05
[M+Na]+
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
Daughter ion
Fix
Parent ion
Vary
Neutral loss
Vary
Neutral gain
Vary
Select-ion monitoring Fix
CID*
Q-3
Vary
Fix
Vary
Vary
Fix
Difference
Vary
Vary
Fix
Fix
Fix
*Collision-induced dissociation (CID) is a reaction region
between quadrupoles
Tandem Mass Spectrometers
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 Scan
2. True Neutral Loss Scan
3. Generate Neutral Gain Scan
4. More Quantitative
5. > Sensitivity for SIM
6. > Dynamic Range
Microbial
Insights, Inc.
CEB
Problem: Rapid Detection/Identification of Microbes
Propose a Sequential High Pressure/Temperature
Extractor Delivers Three Analytes to HPLC/ESI/MS/MS
MeOH
MeOH
CHCl3
PO4-
Pump
CO2
Spe-ed SFE-4
NL
PL
spores
Fraction Collector
N2 blowdown
Auto
sampler
HPLC/ESI/MS/MS