Simultaneous Detection of Low and High Molecular Weight Carbonylated Compounds Derived from Lipid Peroxidation by Electrospray Ionization-Tandem Mass Spectrometry

Reactive oxygen species (ROS) and other oxidative agents such as free radicals can oxidize polyunsaturated fatty acids (PUFA) as well as PUFA in lipids. The oxidation products can undergo consecutive reactions including oxidative cleavages to yield a chemically diverse group of products, such as lipid peroxidation products (LPP). Among them are aldehydes and ketones (“reactive carbonyls”) that are strong electrophiles and thus can readily react with nucleophilic side chains of proteins, which can alter the protein structure, function, cellular distribution, and antigenicity. Here, we report a novel technique to specifically derivatize both low molecular and high molecular weight carbonylated LPP with 7-(diethylamino)­coumarin-3-carbohydrazide (CHH) and analyze all compounds by electrospray ionization-mass spectrometry (ESI-MS) in positive ion mode. CHH-derivatized compounds were identified by specific neutral losses or fragment ions. The fragment ion spectra displayed additional signals that allowed unambiguous identification of the lipid, fatty acids, cleavage sites, and oxidative modifications. Oxidation of docosahexaenoic (DHA, 22:6), arachidonic (AA, 20:4), linoleic (LA, 18:2), and oleic acids (OA, 18:1) yielded 69 aliphatic carbonyls, whose structures were all deduced from the tandem mass spectra. When four phosphatidylcholine (PC) vesicles containing the aforementioned unsaturated fatty acids were oxidized, we were able to deduce the structures of 122 carbonylated compounds from the tandem mass spectra of a single shotgun analysis acquired within 15 min. The high sensitivity (LOD ∼ 1 nmol/L for 4-hydroxy-2-nonenal, HNE) and a linear range of more than 3 orders of magnitude (10 nmol/L to 10 μmol/L for HNE) will allow further studies on complex biological samples including plasma

Carbonylated Plasma Proteins As Potential Biomarkers of Obesity Induced Type 2 Diabetes Mellitus

Protein carbonylation is a common nonenzymatic oxidative post-translational modification, which is often considered as biomarker of oxidative stress. Recent evidence links protein carbonylation also to obesity and type 2 diabetes mellitus (T2DM), though the protein targets of carbonylation in human plasma have not been identified. In this study, we profiled carbonylated proteins in plasma samples obtained from lean individuals and obese patients with or without T2DM. The plasma samples were digested with trypsin, carbonyl groups were derivatized with O-(biotinylcarbazoylmethyl)­hydroxylamine, enriched by avidin affinity chromatography, and analyzed by RPC-MS/MS. Signals of potentially modified peptides were targeted in a second LC-MS/MS analysis to retrieve the peptide sequence and the modified residues. A total of 158 unique carbonylated proteins were identified, of which 52 were detected in plasma samples of all three groups. Interestingly, 36 carbonylated proteins were detected only in obese patients with T2DM, whereas 18 were detected in both nondiabetic groups. The carbonylated proteins originated mostly from liver, plasma, platelet, and endothelium. Functionally, they were mainly involved in cell adhesion, signaling, angiogenesis, and cytoskeletal remodeling. Among the identified carbonylated proteins were several candidates, such as VEGFR-2, MMP-1, argin, MKK4, and compliment C5, already connected before to diabetes, obesity and metabolic diseases

LipidHunter Identifies Phospholipids by High-Throughput Processing of LC-MS and Shotgun Lipidomics Datasets

Lipids are dynamic constituents of biological systems, rapidly responding to any changes in physiological conditions. Thus, there is a large interest in lipid-derived markers for diagnostic and prognostic applications, especially in translational and systems medicine research. As lipid identification remains a bottleneck of modern untargeted lipidomics, we developed LipidHunter, a new open source software for the high-throughput identification of phospholipids in data acquired by LC-MS and shotgun experiments. LipidHunter resembles a workflow of manual spectra annotation. Lipid identification is based on MS/MS data analysis in accordance with defined fragmentation rules for each phospholipid (PL) class. The software tool matches product and neutral loss signals obtained by collision-induced dissociation to a user-defined white list of fatty acid residues and PL class-specific fragments. The identified signals are tested against elemental composition and bulk identification provided via LIPID MAPS search. Furthermore, LipidHunter provides information-rich tabular and graphical reports allowing to trace back key identification steps and perform data quality control. Thereby, 202 discrete lipid species were identified in lipid extracts from rat primary cardiomyocytes treated with a peroxynitrite donor. Their relative quantification allowed the monitoring of dynamic reconfiguration of the cellular lipidome in response to mild nitroxidative stress. LipidHunter is available free for download at https://bitbucket.org/SysMedOs/lipidhunter

Recent Advances on Mass Spectrometry Analysis of Nitrated Phospholipids

In recent years, there has been an increasing interest in nitro fatty acids (NO₂-FA) as signaling molecules formed under nitroxidative stress. NO₂-FA were detected <i>in vivo</i> in a free form, although it is assumed that they may also be esterified to phospholipids (PL). Nevertheless, insufficient discussion about the nature, origin, or role of nitro phospholipids (NO₂-PL) was reported up to now. The aim of this study was to develop a mass spectrometry (MS) based approach which allows identifying nitroalkenes derivatives of three major PL classes found in living systems: phosphatidylcholines (PCs), phosphatidylethanolamine (PEs), and phosphatidylserines (PSs). NO₂-PLs were generated by NO₂BF₄ in hydrophobic environment, mimicking biological systems. The NO₂-PLs were then detected by electrospray ionization (ESI-MS) and ESI-MS coupled to hydrophilic interaction liquid chromatography (HILIC). Identified NO₂-PLs were further analyzed by tandem MS in positive (as [M + H]⁺ ions for all PL classes) and negative-ion mode (as [M – H]⁻ ions for PEs and PSs and [M + OAc]⁻ ions for PCs). Typical MS/MS fragmentation pattern of all NO₂-PL included a neutral loss of HNO₂, product ions arising from the combined loss of polar headgroup and HNO₂, [NO₂-FA + H]⁺ and [NO₂-FA – H]⁻ product ions, and cleavages on the fatty acid backbone near the nitro group, allowing its localization within the FA akyl chain. Developed MS method was used to identify NO₂-PL in cardiac mitochondria from a well-characterized animal model of type 1 diabetes mellitus. We identified nine NO₂-PCs and one NO₂-PE species. The physiological relevance of these findings is still unknown

Additional file 1: of Differential gene expression in response to Fusarium oxysporum infection in resistant and susceptible genotypes of flax (Linum usitatissimum L.)

Genes for which expression was specifically changed in resistant genotypes of flax in response to Fusarium oxysporum infection. (XLSX 9584 kb

Additional file 2: of Differential gene expression in response to Fusarium oxysporum infection in resistant and susceptible genotypes of flax (Linum usitatissimum L.)

Expression profiles of genes for selected gene ontology (GO) terms in susceptible and resistant flax genotypes in response to Fusarium oxysporum infection. (XLSX 861 kb

Additional file 1: Table S1. of Differential expression of alternatively spliced transcripts related to energy metabolism in colorectal cancer

Differential expression profiles of genes and transcripts in colon adenocarcinomas compared to matched normal tissues (TCGA data analyzed by CrossHub). (XLSX 43 MB

Additional file 3: Table S3. of HK3 overexpression associated with epithelial-mesenchymal transition in colorectal cancer

(XLS 34 kb

Additional file 1: Table S1. of HK3 overexpression associated with epithelial-mesenchymal transition in colorectal cancer

(XLS 393 kb

Additional file 5: of Exome analysis of carotid body tumor

Total number of deleterious somatic mutations normalized by gene length in 52 CBT samples. (XLSX 132 kb