Lipid extraction, quantification, and characterization
This protocol is extracted from research article:
Escherichia coli as a platform for the study of phosphoinositide biology
Sci Adv, Mar 27, 2019; DOI: 10.1126/sciadv.aat4872

For lipid extraction, a volume of culture equivalent to 10 ml of OD600 1.0 was pelleted, and phospholipids were extracted using a variant of the Bligh-Dyer protocol (54). Briefly, the pellet was resuspended in 420 μl of water, and 1250 μl of methanol and 625 μl of chloroform were added in that order. This mix was vortexed, and another 625 μl of chloroform was added. After vortexing of the new mix, 600 μl of water was added and the mix was vortexed again. Last, 25 μl of HCl was added to acidify the mix, and after repeat vortexing, the mix was centrifuged at 150 rcf for 5 min. The bottom (organic) phase was then dried under argon and resuspended in a chloroform/methanol 20:9 mix. The extracted lipids were then stored at −20°C until measurements were carried out, 1 to 10 days after the extraction. PI4P and PIP2 abundances were measured using a competitive ELISA commercially available from Echelon Biosciences. On the day of the ELISA tests, an aliquot of 6 μl or less of the extracted lipids was dried and resuspended according to the ELISA protocol. A minor level of signal is always detected in these ELISA tests, even for controls, indicating some background level of noise when using phospholipids extracted from E. coli. These ELISAs proved to be problematic, in many cases showing only a background signal for all samples. Repeating the ELISA test on a different day with the same extracts and protocol was the only way to successfully measure the phosphoinositides. We therefore only report data from tests in which the samples show variation depending on the construct used; this means that unsuccessful ELISA tests, for which no signal was detected above background for any samples, were not used. While this could potentially introduce a sampling bias by selecting only for positive results, the samples measured successfully are the same as those that produced no signal on the failed test days; thus, there is no risk of bias in the results reported. Troubleshooting the assay with the vendor’s suggestions did not help; the presumption is that differences in room temperature and/or humidity alter the first step of the ELISA process in which the phospholipids are resuspended in an aqueous solution; thus, in failed tests, the presumption is that the lipid never resuspended successfully. The test is reported by the vendor to be robust, and it is possible that the shorter lipid tails produced in bacteria are responsible for this erratic behavior. Initially, we used a strip lipid protein overlay for PI4P detection (also from Echelon Biosciences), which worked reliably, but this product was discontinued and we could not reproduce its signal-to-noise ratios even when acquiring all the reagents from the same company.

To characterize the phosphoinositides produced in the bacteria, we used Avanti Polar Lipids’ analytical services to get a first view of the different phospholipid classes. For this analysis, we sent dry lipid extracts processed as described earlier and dried under argon. At the vendor’s facilities, the lipids were re-extracted to remove impurities, and this extract was then analyzed by mass spectrometry with the appropriate standards for quantification. This analysis, however, was only performed for PI, since phosphorylated phosphoinositides are not easily modified to be amenable for mass spectrometry. We also used the services of ATK Innovation, Analytics and Discovery (North Bend, WA), which performed a comprehensive mass spectrometry analysis of our samples. Phosphoinositides were measured in acidic extracts both with and without prior neutral extraction using a chemical modification of the phosphate group in the phosphorylated phosphoinositides as described in the literature (55, 56). Neutral lipids were measured using methods adapted from those of Hines et al. (57). Internal standards obtained from Avanti Polar Lipids (Alabaster, AL) added at the beginning of the extraction protocol paired with standard curves in comparable samples were used to normalize data, controlling for extraction bias and matrix effects. We used the data obtained with this quantification to perform a linear conversion on the ELISA data to correct for extraction and protocol losses.

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