Mass spectrometry-guided molecular omics approaches for the study of lipid metabolism connected with the peroxisome/PPAR system

Abstract

Multi-layered molecular (i.e. lipids, metabolites, and proteins) read-outs can provide deeper insights into biochemical cascades and disease mechanisms. Mass spectrometry (MS) has emerged as an invaluable and elemental analytical tool to study these molecular components in biological systems. Peroxisomes are small, omnipresent subcellular organelles that perform a wide range of functions in human health and disease. They participate in numerous degradative and biosynthetic processes, including lipid metabolism. Peroxisomal dysfunction leads to devastating genetic human disorders, namely peroxisomal biogenesis disorders (PBDs) and single peroxisomal enzyme deficiencies (PEDs). PBDs arise from mutations in Pex genes that encode peroxisomal biogenesis proteins (also named peroxins, PEX). The PEX11 protein family (α, β, and γ isoforms) regulates fission and proliferation of peroxisomes. However, detailed physiological and molecular functions of PEX11 protein isoforms, pathological and metabolic consequences owing to their defects are still being discovered. In this thesis, MS-guided molecular omics (i.e. lipidomics, metabolomics, and proteomics) workflows were developed and employed to ascertain the molecular compositional changes occurring due to Pex11α and Pex11β deficiencies, as well as during the postnatal lung development. The cutting-edge analytical technologies used for this purpose were ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS), atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI), and direct flow injection electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the first article, altered molecular species and metabolic pathways were depicted in serum, liver, and heart tissues of untreated Pex11α knockout adult mice in comparison to the wild type controls. Primarily, a single-step liquid-liquid extraction (MTBE/MeOH/H2O) method was utilized and optimized for the parallel extraction of lipids (upper organic phase), polar metabolites (lower aqueous phase), and proteins (sediment pellet) from the same and limited amount of biological specimens. Later, the dried lipid, polar metabolite, and protein extracts were quantitatively analyzed using a reversed-phase UHPLC-MS/MS system individually in positive- and negative-ion mode. In addition, extensive data filtering and manual curation strategies were implemented and high-confidence molecular annotations were reported by removing/minimizing the false positives. Furthermore, comprehensive specimen-specific multiple molecular species and related metabolic pathway changes were uncovered in distinct biological specimens of Pex11α knockout mice. In the second article, MALDI MS imaging was used to characterize the full lipidome of late fetal mouse lungs at day 19 of gestation (E19) followed by semi-quantitative analysis of lipids in E19 WT and Pex11β knockout mice lung tissue sections. Sample preparation protocol including tissue handling, sectioning of E19 mouse lungs without embedding material, homogenous deposition of matrices, instrumental and data processing framework were optimized. MS imaging experiments were carried out at a high resolution in mass (140,000 @ m/z 200) and space (10 µm per pixel) using an AP-SMALDI10 ion source. E19 mouse lung full lipidome was characterized based on accurate mass (≤ 2 ppm) and on-tissue tandem mass spectrometry experiments in both positive- and negative-ion mode. Additionally, data handling and analysis strategies including different normalization approaches were tested and optimized for direct comparison of relative signal intensities of lipids and endogenous metabolites among different tissue sections. Furthermore, the developed MSI workflow was employed and illustrated the molecular changes in Pex11β knockout mouse lung tissues in comparison to the E19 WT controls. In the third article, direct flow injection quantitative lipidomic analysis (also called shotgun lipidomics) was carried out to monitor the compositional changes in mouse lung lipidome during the postnatal development process, from birth to adulthood. Lipids were extracted from whole lung tissue homogenates of newborn (P1), 15-day-old (P15), and 12-week-old (P84) adult mice using CHCl3/MeOH/H2O liquid-liquid extraction (Bligh and Dyer method). Then, the extracted lipids were quantified using electrospray ionization tandem mass spectrometry in positive-ion mode. Overall, an extensive quantitative (molar abundances) lipidome of mouse lung and significant stage-specific alterations of lipid classes and individual lipid molecular species were presented during the postnatal pulmonary development process.