Protocols – MASSTRPLAN

Protocol descriptions

Optimized reverse phase chromatography for lipidomics and oxidized phospholipids – Protocol#2

Analysis of lipid and especially lipid peroxidation products (LPPs) can be challenging from many different points of view. Natural lipidomes may be extremely complex with lipids belonging to diverse group of compounds and different for physico-chemical properties. The high incidence of isomeric and isobaric compounds, molecules with same molecular weight or very similar molecular weight but different structure, complicates lipidomics analysis even more. The high dynamic range of lipid concentration in biological matrices leads to the underestimation of low abundant lipids. Shotgun and LC–MS based data-dependent acquisition (DDA) methods are the most common techniques used in untargeted mass spectrometry (MS)-based lipidomics. Shotgun lipidomics is a robust and high-throughput method for lipid analysis. It allows identification and quantification of many lipids in a short period of time. However, identification on the interpretation of the MS/MS spectra may be very challenging for isomeric and isobaric species. Liquid chromatography coupled on-line to mass spectrometry allows high-throughput characterization of lipids, reducing suppression and matrix effects, increasing peak capacity of the analytical platform, and allowing chromatographic enrichment, which may help to resolve potential isomers or isobars. Reverse phase liquid chromatography (RPLC) is particularly suitable for the analysis of lipid peroxidation products as it is possible to separate isomers and positional isomers and importantly, they can also be separated from isobaric native species. The protocols have tested for lipidomics of plasma, and on oxidized phospholipids prepared in vitro by autoxidation. For the full protocol (PDF): click

Diagnostic ions for small aldehyde modifications of proteins – Protocol#4

Small reactive aldehydes derived from lipid peroxidation or glycoxidation can form adducts on protects, a process called lipoxidation. Lipoxidation adducts have been found in several inflammatory diseases, including atherosclerosis and Alzheimer’s disease. The detection and analysis of these adducts in biological samples is challenging because of their low abundance and the difficulty of interpreting mass spectrometry data from proteomic analysis. LC-MS is nowadays the technique of choice for the identification of protein-lipid adducts, since it can detect the mass shift caused by the adducts, and when used in tandem mode allows the modified amino acid to be located in the protein sequence. In complex samples thus is still challenging owing to many false positive identifications, as well as the possibility of failing to detect the lower abundance modified peptides in the presence of high abundance unmodified ones. To help overcome this, the information on the fragmentation patterns of aldehyde-protein adducts can be used to identify reporter ions, which are fragment ions characteristic and specific for each modification, rather than for the peptide sequence. These reporter ions can be used to add extra confidence to the identification of modifications in vivo, or search more specifically for the modification by semi-targeted mass spectrometry approaches, e.g. precursor ion scanning (PIS) or neutral loss scanning (NLS), thus simplifying the analysis.
This protocol details the detection of diagnostic ions for several small aldehyde adducts on model proteins lysozyme, human serum albumin and pyruvate kinase, based on our methods reported in Afonso et al. [1] and Sousa et al. [2]. For the full protocol (PDF): click

Reversed-phase LC-MS and MS/MS analysis of nitroxidative modifications in Cardiolipin – Protocol#5

Nitroxidative modifications are of increasing interest owing to the understanding that nitrating and oxidizing conditions occur in inflammation and several associated diseases, such as cardiovascular disease and neurodegenerative diseases, through formation of the highly reactive compound peroxynitrite (ONOO-) and radical nitroxy species. These have long been known to attack unsaturated fatty acids, leading to the formation of nitrated fatty acids, well known endogenous lipid mediators. More recently, modification of phospholipids by these reactive nitrogen species was also reported, leading to formation of nitrated and nitroxy products that have a variety of biological activities. However, previously the formation of such products on the mitochondrial lipid cardiolipin had not be studied.
This protocol details how to obtain nitroxidation products of cardiolipin using an in vitro model of nitroxidation with NO2BF4, followed by methods for structural characterization by reversed-phase C30 liquid chromatography – high-resolution mass spectrometry. The protocol is based on the methods and data that we have reported in [1]. For the full protocol (PDF): click