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Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories

Wu, C.-H. and Kong, L. and Bialecka-Fornal, M. and Park, S. and Thompson, A. L. and Kulkarni, G. and Conway, S. J. and Newman, D. K. (2015) Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories. Geobiology, 13 (4). pp. 391-407. ISSN 1472-4677. PMCID PMC4676935.

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Hopanoids are steroid-like lipids from the isoprenoid family that are produced primarily by bacteria. Hopanes, molecular fossils of hopanoids, offer the potential to provide insight into environmental transitions on the early Earth, if their sources and biological functions can be constrained. Semiquantitative methods for mass spectrometric analysis of hopanoids from cultures and environmental samples have been developed in the last two decades. However, the structural diversity of hopanoids, and possible variability in their ionization efficiencies on different instruments, have thus far precluded robust quantification and hindered comparison of results between laboratories. These ionization inconsistencies give rise to the need to calibrate individual instruments with purified hopanoids to reliably quantify hopanoids. Here, we present new approaches to obtain both purified and synthetic quantification standards. We optimized 2-methylhopanoid production in Rhodopseudomonas palustris TIE-1 and purified 2Me-diplopterol, 2Me-bacteriohopanetetrol (2Me-BHT), and their unmethylated species (diplopterol and BHT). We found that 2-methylation decreases the signal intensity of diplopterol between 2 and 34% depending on the instrument used to detect it, but decreases the BHT signal less than 5%. In addition, 2Me-diplopterol produces 10× higher ion counts than equivalent quantities of 2Me-BHT. Similar deviations were also observed using a flame ionization detector for signal quantification in GC. In LC-MS, however, 2Me-BHT produces 11× higher ion counts than 2Me-diplopterol but only 1.2× higher ion counts than the sterol standard pregnane acetate. To further improve quantification, we synthesized tetradeuterated (D_4) diplopterol, a precursor for a variety of hopanoids. LC-MS analysis on a mixture of (D4)-diplopterol and phospholipids showed that under the influence of co-eluted phospholipids, the D_4-diplopterol internal standard quantifies diplopterol more accurately than external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples.

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URLURL TypeDescription Information CentralArticle
Newman, D. K.0000-0003-1647-1918
Additional Information:© 2015 The Authors. Geobiology Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Received 31 August 2014; accepted 22 February 2015. Article first published online: 11 Apr 2015. We thank members of Newman and Conway laboratories for critical comments on the manuscript. We thank Dr. Kun-Liang Wu, Dr. Chi-Li Chen, and Allen Lee for help with hopanoid purification, and Lichun Zhang and Dr. Fenfang Wu for assistance with GC-MS. We thank Dr. Nathan Dalleska for help with LC-MS. The UPLC-TOF MS equipment in the California Institute of Technology’s Environmental Analysis Center was used in the work. We also thank Collin Sparrow for FI measurements. This work was supported by grants from NASA (NNX12AD93G), the National Science Foundation (1224158), and the Howard Hughes Medical Institute (HHMI) to DKN and grants (EP/K000888/1; EP/L000067/1) from the Engineering and Physical Sciences Research Council to SJC. DKN is an HHMI Investigator. SJC thanks St Hugh’s College, Oxford, for research support.
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Engineering and Physical Sciences Research Council (EPSRC)EP/K000888/1
Engineering and Physical Sciences Research Council (EPSRC)EP/L000067/1
St Hugh’s College, OxfordUNSPECIFIED
Issue or Number:4
PubMed Central ID:PMC4676935
Record Number:CaltechAUTHORS:20150421-104932645
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Official Citation:Wu, C.-H., Kong, L., Bialecka-Fornal, M., Park, S., Thompson, A. L., Kulkarni, G., Conway, S. J. and Newman, D. K. (2015), Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories. Geobiology, 13: 391–407. doi: 10.1111/gbi.12132
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:56813
Deposited By: Ruth Sustaita
Deposited On:21 Apr 2015 18:19
Last Modified:03 Oct 2019 08:17

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