Comprehensive and Quantitative Analysis of Polyphosphoinositide Species by Shotgun Lipidomics Revealed Their Alterations in db/db Mouse Brain

Abstract

Polyphosphoinositides (PPI) play crucial roles in cellular signaling and functions. However, comprehensively determining the changed levels of these species during different cellular processes has faced difficulties. Herein, we applied a novel methylation pattern recognition and simulation approach, and we exploited newly derived fragmentation patterns of methylated PPI species for comprehensive analysis of PPI species including phosphate position(s) and fatty acyl chains capable of circumpassing previous limitations. The developed method was applied for quantitative analysis of PPI species present in diabetic mouse cortex and liver, and it allowed us to unravel the marked reduction of PPI levels in brain cortices of db/db mice for the first time. Taken together, we developed a powerful and high-throughput method for comprehensive analysis of PPI species, which should greatly contribute to the elucidation of PPI biology under different disease states.

Fatty-acyl chain profiles of cellular phosphoinositides

“…Subsequently, and key to our method was to promote the formation of sodiated adducts. Hsu and coworkers had added LiOH to the mixture for infusion into the ESI source of a Finnigan TSQ-7000 triple stage quadrupole spectrometer to generate lithiated adducts of phosphatidylcholine and later extended this technique to include phosphatidylethanolamines [35, 36] and by others to polyphosphoinositides [37]. The post-column infusion option of the Waters Xevo TQ bypassed the need for adding sodium to the mobile phase.…”

“…While our paper was under review, a new mass spectrometry method for polyphosphoinositides was published [37]. The technique was shotgun lipidomics that did not use liquid chromatography but rather multiple sequential extractions, methyl derivatization, and 5-min continuous infusion into a nanospray triple-quadrupole mass spectrometer running both in positive ion mode and in negative ion mode.…”

Understanding phosphoinositides: rare, dynamic, and essential membrane phospholipids

“…For PPI lipids, the negative phosphate groups in the sample need to be methylated to make them neutral [70]. The analysis of ionized fragments yields the atomic mass, readily making possible a determination of the number of phosphates, the total chain length of attached fatty acid side chains, and their degree of unsaturation [71–73]. With care for sensitivity, more than a dozen different fatty acid side-chain combinations are readily resolved for each major PPI class.…”

Sensitive analysis of fatty acid esters of hydroxy fatty acids in biological lipid extracts by shotgun lipidomics after one-step derivatization

“…Unfortunately, many FAHFA species have not been commercially available. Therefore, an alternative method through trial and error in combination with ramping collision energy in analysis of biological samples with one IS was employed to determine the appropriate collision energy to balance the differential fragmentation effects as previously described [26,27].…”