Improved extraction and complete mass spectral characterization of steroidal alkaloids from Veratrum californicum.

Four steroidal alkaloids extracted from the roots and rhizomes of Veratrum californicum were separated by high performance liquid chromatography (HPLC) and identified using high resolution electrospray ionization time of flight tandem mass spectrometry (ESI-TOF-MS/MS) as veratrosine, cycloposine, veratramine, and cyclopamine. This paper compares ethanol and benzene as extraction solvents, HPLC solvent conditions leading to good separation of steroidal alkaloids, and MS/MS fragmentation patterns for the four steroidal alkaloids which have been released to the public database MassBank.jp.

The frequency of 1,4-benzoquinone-lysine adducts in cytochrome c correlate with defects in apoptosome activation.

Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species.

Identification of chemical-adducted proteins in urine by multi-dimensional protein identification technology (LC/LC-MS/MS).

Recent technological advancements in mass spectrometry facilitate the detection of chemical-induced posttranslational modifications (PTMs) that may alter cell signaling pathways or alter the structure and function of the modified proteins. To identify such protein adducts (Kleiner et al., Chem Res Toxicol 11:1283-1290, 1998), multi-dimensional protein identification technology (MuDPIT) has been utilized.

One-dimensional western blotting coupled to LC-MS/MS analysis to identify chemical-adducted proteins in rat urine.

The environmental toxicant hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis via a combination of redox cycling and the covalent adduction of proteins within the S₃ segment of the renal proximal tubules in the outer stripe of the outer medulla (OSOM). Following administration of 2-(glutathion-S-yl)HQ (MGHQ) (400 μmol/kg, i.v.

Utilization of LC-MS/MS analyses to identify site-specific chemical protein adducts in vitro.

Biologically reactive intermediates are formed following metabolism of xenobiotics, and during normal oxidative metabolism. These reactive species are electrophilic in nature and are capable of forming stable adducts with target proteins. These covalent protein modifications can initiate processes that lead to acute tissue injury or chronic disease.

Utilization of MALDI-TOF to determine chemical-protein adduct formation in vitro.

Biological reactive intermediates can be created via metabolism of xenobiotics during the process of chemical elimination. They can also be formed as by-products of cellular metabolism, which produces reactive oxygen and nitrogen species. These reactive intermediates tend to be electrophilic in nature, which enables them to interact with tissue macromolecules, disrupting cellular signaling processes and often producing acute and chronic toxicities.

Protein electrophile-binding motifs: lysine-rich proteins are preferential targets of quinones.

Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics, and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. It is likely that topological, chemical, and physical features combine to determine which proteins become targets for chemical adduction. Chemical-induced post-translational modification of certain critical proteins causes a change in structure/function that contributes to the toxicological response to chemical exposure.

Quinone electrophiles selectively adduct "electrophile binding motifs" within cytochrome c.

Electrophiles generated endogenously, or via the metabolic bioactivation of drugs and other environmental chemicals, are capable of binding to a variety of nucleophilic sites within proteins. Factors that determine site selective susceptibility to electrophile-mediated post-translational modifications, and the consequences of such alterations, remain largely unknown. To identify and characterize chemical-mediated protein adducts, electrophiles with known toxicity were utilized.