12 research outputs found
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 (NO<sub>2</sub>-FA) as signaling molecules formed under
nitroxidative stress. NO<sub>2</sub>-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 (NO<sub>2</sub>-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).
NO<sub>2</sub>-PLs were generated by NO<sub>2</sub>BF<sub>4</sub> in
hydrophobic environment, mimicking biological systems. The NO<sub>2</sub>-PLs were then detected by electrospray ionization (ESI-MS)
and ESI-MS coupled to hydrophilic interaction liquid chromatography
(HILIC). Identified NO<sub>2</sub>-PLs were further analyzed by tandem
MS in positive (as [M + H]<sup>+</sup> ions for all PL classes) and
negative-ion mode (as [M – H]<sup>−</sup> ions for PEs
and PSs and [M + OAc]<sup>−</sup> ions for PCs). Typical MS/MS
fragmentation pattern of all NO<sub>2</sub>-PL included a neutral
loss of HNO<sub>2</sub>, product ions arising from the combined loss
of polar headgroup and HNO<sub>2</sub>, [NO<sub>2</sub>-FA + H]<sup>+</sup> and [NO<sub>2</sub>-FA – H]<sup>−</sup> 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 NO<sub>2</sub>-PL in cardiac mitochondria from
a well-characterized animal model of type 1 diabetes mellitus. We
identified nine NO<sub>2</sub>-PCs and one NO<sub>2</sub>-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 9584Â 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 861Â 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 132Â kb