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Expanded Utility of Signature Lipid Biomarker Analysis for

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.

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Expanded Utility of Signature Lipid Biomarker Analysis for

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  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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 frompure culture and environmental samples using pressurized accelerated hot solvent extraction. J. Microbial Methods 31: 19-27(1997) CEB Microbial Insights, Inc.

  9. ESI (cone voltage) Q-1 CID Q-3 FRAGMENTATION with ESI/MS/MS

  10. Respiratory Ubiquinone (UQ) Gram-negative Bacteria with Oxygen as terminal acceptor LOQ = 225 femtomole/uL, LOD = 75 femtomole/uL ~ 100E. coli Isocratic 95.5/4.5 % methanol/aqueous 1 mM ammonium acetate Q7 Q10 Q6 197 m/z

  11. Pyridinium Derivative of 1, 2 Dipalmitin [M+92-109]+ M = mass of original Diglyceride LOD ~100 attomoles/ uL [M+92]+

  12. 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

  13. 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

  14. 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 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.

  15. 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 747scanned m/z 110-990 Note 50X > sensitivity SIM additional 5x > sensitivity ~ 250X

  16. 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 16:0 255 271 Unusual 12C 17:0 (269) + 2 13C  cy17:0 267 284 12C 18:0 (283) + 13C 18:1 281 29912C 20:6 , 12C 19:0 with 2 13C  19:1 295 314 12C 21:5 (315), 12C 21:6 (313)  13C bacteria added  No 13C bacteria added

  17. Archaebacterial Tetraether Lipid FW 1640.4 In sim LOQ ~ 50 ppb ES+ [M-2H+Na+K]+ [M+H]+

  18. 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 [M+Na]+

  19. 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

  20. 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

  21. Problem: Rapid Detection/Identification of Microbes Propose a Sequential High Pressure/Temperature Extractor Delivers Three Analytes to HPLC/ESI/MS/MS

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