Inactivation and Extraction of Bacterial Spores for Systems Biological Analysis

Powell, Bradford S.; Cybulski, Robert J.

doi:10.1007/978-94-007-0828-0_46

Cited by 1 publication

(1 citation statement)

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“…(ii) Sample homogenization: To achieve reproducible and robust lipid extraction, sample preparation must ensure proper homogenization and, depending on study goals, must consistently lyse cells (eukaryotic, eukaryotic and prokaryotic, or neither). The PCT protocol here in conjunction with hexafluoroisopropanol lyses both eukaryotic and prokaryotic cells, as well as most prokaryotic spores. , (iii) Solvent choice: The solvent must address the complexity of fecal samples (e.g., mixed polar, nonpolar and neutral lipids in a matrix of water-soluble and insoluble material). Both solvent chemistries were tested with and without PCT, on three dilutions of starting material.…”

Section: Resultsmentioning

confidence: 99%

Method Development for Fecal Lipidomics Profiling

et al. 2012

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Robust methodologies for the analysis of fecal material will facilitate the understanding of gut (patho)physiology, its role(s) in health and disease and help improve care for individual patients, especially high-risk populations such as premature infants. Because lipidomics offers a biologically and analytically attractive approach, we developed a simple, sensitive, and quantitatively precise method for profiling intact lipids in fecal material. The method utilizes two separate, complementary extraction chemistries, dichloromethane (DCM) and a methyl tert-butyl ether/hexafluoroisopropanol (MTBE) mixture, alone or with high pressure cycling. Extracts were assessed by liquid chromatography-high resolution mass spectrometry-based profiling with all ion higher energy collisional dissociation fragmentation in both positive and negative ionization modes. This approach provides both class-specific and lipid-specific fragments, enhancing lipid characterization. Solvents preferentially extracted lipids based on hydrophobicity. More polar species preferred MTBE; more hydrophobic compounds preferred DCM. Pressure cycling differentially increased the yield of some lipids. The platform enabled analysis of >500 intact lipophillic species with over 300 lipids spanning 6 LIPID MAPS categories identified in the fecal matter from premature infants. No previous report exists that provides these data; thus, this study represents a new paradigm for assessing nutritional health, inflammation, and infectious disease in vulnerable populations.

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Section: Resultsmentioning

confidence: 99%