Analysis of Lipid Experiments (ALEX): A Software Framework for Analysis of High-Resolution Shotgun Lipidomics Data

Abstract

Global lipidomics analysis across large sample sizes produces high-content datasets that require dedicated software tools supporting lipid identification and quantification, efficient data management and lipidome visualization. Here we present a novel software-based platform for streamlined data processing, management and visualization of shotgun lipidomics data acquired using high-resolution Orbitrap mass spectrometry. The platform features the ALEX framework designed for automated identification and export of lipid species intensity directly from proprietary mass spectral data files, and an auxiliary workflow using database exploration tools for integration of sample information, computation of lipid abundance and lipidome visualization. A key feature of the platform is the organization of lipidomics data in ”database table format” which provides the user with an unsurpassed flexibility for rapid lipidome navigation using selected features within the dataset. To demonstrate the efficacy of the platform, we present a comparative neurolipidomics study of cerebellum, hippocampus and somatosensory barrel cortex (S1BF) from wild-type and knockout mice devoid of the putative lipid phosphate phosphatase PRG-1 (plasticity related gene-1). The presented framework is generic, extendable to processing and integration of other lipidomic data structures, can be interfaced with post-processing protocols supporting statistical testing and multivariate analysis, and can serve as an avenue for disseminating lipidomics data within the scientific community. The ALEX software is available at www.msLipidomics.info.

Comprehensive Lipidome Analysis by Shotgun Lipidomics on a Hybrid Quadrupole-Orbitrap-Linear Ion Trap Mass Spectrometer

“…Comparing the composition of lipid species, however, showed more pronounced differences; for example, the species PC 32:0, PC 36:4, and PC 38:4 were enriched in hippocampus compared with cerebellum, and the species PC 38:6 and PC 40:6 were enriched in cerebellum compared with hippocampus ( Figure 5b). Notably, the obtained composition of lipid classes and lipid species in cerebellum and hippocampus is consistent with previous reports on mouse brain lipid composition [32,50]. Based on these results, we conclude that the MS ALL routine with FTMS analysis using a resolution setting of 450,000 affords reproducible and comprehensive lipidome quantification.…”

“…Lipid species detected by MS ALL using high resolution FTMS analysis were identified and quantified using ALEX software as previously described [32]. In short, lipid species detected by FTMS and annotated by sum composition were quantified by normalizing their intensity to the intensity of an internal lipid standard of identical lipid class and multiplying with the spike amount of the internal lipid standard and the type I isotope correction factor [32][33][34] [32] and further processed for data visualization, filtering, and molecular glycerophospholipid species identification using Tableau Desktop (Tableau Software).…”

“…In short, lipid species detected by FTMS and annotated by sum composition were quantified by normalizing their intensity to the intensity of an internal lipid standard of identical lipid class and multiplying with the spike amount of the internal lipid standard and the type I isotope correction factor [32][33][34] [32] and further processed for data visualization, filtering, and molecular glycerophospholipid species identification using Tableau Desktop (Tableau Software). An intensity filter was applied for identification requiring that ions detected by FTMS, FTMS 2 and ITMS 3 should have intensity higher than 2000, 500 and 1 count, respectively (values determined by manual assessment of spectral quality), and be detected in at least half of all sample injections.…”

“…For quantitative lipidome analysis, we processed the high resolution FTMS data using ALEX software [32]. ALEX software affords identification of lipid species detected by high resolution FTMS analysis and compiles output data in a database table format.…”

“…123 was designed to compile output data in database table format [32], which allows tracking all experimental data across large sample sets (e.g., polarity, ion activation, ion detection, measured m/z values of all fragment ion across all samples) and subsequent data filtering, quality control, and visualization using Tableau Desktop ( Figure 5 and S3 in Supporting Information).…”