Human Brain Lipidomics: Utilities of Chloride Adducts in Flow Injection Analysis.

Paul L Wood,Jon H Scarborough,Randall L Woltjer,Beatrix Dudzik,John E Cebak,Kathleen A Hauther,Dustin J Craney

doi:10.3390/life11050403

Abstract

Ceramides have been implicated in a number of disease processes. However, current means of evaluation with flow infusion analysis (FIA) have been limited primarily due to poor sensitivity within our high-resolution mass spectrometry lipidomics analytical platform. To circumvent this deficiency, we investigated the potential of chloride adducts as an alternative method to improve sensitivity with electrospray ionization. Chloride adducts of ceramides and ceramide subfamilies provided 2- to 50-fold increases in sensitivity both with analytical standards and biological samples. Chloride adducts of a number of other lipids with reactive hydroxy groups were also enhanced. For example, monogalactosyl diacylglycerols (MGDGs), extracted from frontal lobe cortical gray and subcortical white matter of cognitively intact subjects, were not detected as ammonium adducts but were readily detected as chloride adducts. Hydroxy lipids demonstrate a high level of specificity in that phosphoglycerols and phosphoinositols do not form chloride adducts. In the case of choline glycerophospholipids, the fatty acid substituents of these lipids could be monitored by MS2 of the chloride adducts. Monitoring the chloride adducts of a number of key lipids offers enhanced sensitivity and specificity with FIA. In the case of glycerophosphocholines, the chloride adducts also allow determination of fatty acid substituents. The chloride adducts of lipids possessing electrophilic hydrogens of hydroxyl groups provide significant increases in sensitivity. In the case of glycerophosphocholines, chloride attachment to the quaternary ammonium group generates a dominant anion, which provides the identities of the fatty acid substituents under MS2 conditions.

Highlights

While ceramide analysis was the focus of our method development, we investigated other lipids to define the scope of chloride adducts and their utility in characterizing other lipids
The repeatability of chloride adduct analysis was evaluated by 10 repeated injections of a brain gray matter (GM) extract daily for 5 days
Our analyses demonstrated that the relative levels of almost all ceramides, hydroxyceramides, hexosylceramides, hexosylhydroxyceramides, phytoceramides, and ceramide phosphoethanolamines were higher in the white matter (WM) than the GM in the human frontal cortex (Figure 1)

Summary

Introduction

Ceramides have come under increasing scrutiny as potential biomarkers in a number of diseases [1]. Lack of consistency in reported alterations of ceramide levels probably relates to both the selection of analytical methods and the quality of the biological specimens. In the case of Alzheimer’s disease brain samples, large increases in ceramide levels have been reported with triple quadrupole-electrospray ionization (ESI)-mass spectrometry [2,3]. Triple quadrupole-ion trap ESI and Orbitap-ESI methods have reported small or no increases in cortical ceramides [4,5,6,7], suggesting that high-resolution mass spectrometry provides more accurate measurements of these lipids. Our flow infusion ESI lipidomics analytical platform [5,8] has been limited by weak [M+H]+ ,

Methods

Results

Discussion

Conclusion