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

PLFA (phospholipid fatty acid) excellent ~x 102-103 ppm Cr with (PLFA).

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

Why not Universally utilized?

1. Requires 8 hr extraction with ultrapure solvents [emulsions]. 2. Ultra clean glassware [incinerated 450oC]. 3. Fractionation of Polar Lipids4. 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 acceptorDiglyceride ~ cell lysisArchea ~ methanogensLipid ornithine ~ bioavailable phosphateLysyl-phosphatidyl glycerol ~ low pHPoly beta-hydroxy alkanoate ~ unbalanced growth

3. Increased Sensitivity and Specificity ESI/MS/MS

Signature Lipid Biomarker Analysis

Lyophilized Soil Fractions, Pipe Biofilm

SFECO2 1. Neutral Lipids

UQ isoprenologues

Derivatize –N-methyl pyridyl Diglycerides Sterols Ergostrerol Cholesterol

ESE Chloroform.methanol

2. Polar Lipids

Transesterify

PLFA

CG/MS

Intact Lipids

HPLC/ESI/MS/MS

Phospholipids PG, PE, PC, Cl, & sn1 sn2 FAAmino Acid PGOrnithine lipidArchea ether lipidsPlamalogens

PHAThansesterify & Derivatize N-methyl pyridyl

3. In-situ Derivatize in SFECO2

2,6 DPA (Spores)

LPS-Amide OH FA

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

*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

[2 cycles 80oC, 1200 psi, 20 min] vs B&D = 8 -14 Hours

CEBMicrobial Insights, Inc.

ESI (cone voltage) Q-1 CID Q-3

FRAGMENTATION with ESI/MS/MS

Q6

Q7Q10

O

O

H3OC

H3OC

CH3

]H

n

197 m/z

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

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

16:0 255 271 Unusual 12C 17:0 (269) + 2 13C cy17:0 267 284 12C 18:0 (283) + 13C

18:1 281 299 12C 20:6 , 12C 19:0 with 2 13C 19:1 295 314 12C 21:5 (315), 12C 21:6 (313)

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

CO2

Pump

N2 blowdownAutosampler

HPLC/ESI/MS/MS

Fraction Collector

Spe-ed SFE-4 NL

PL

spores

MeOHMeOHCHCl3

PO4-