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 reported Soxhlet extraction method nearly triples the recovery of steroidal alkaloids from V. californicum.

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. Site-specific BQ-lysine adducts are found on residues in cytochrome c that are necessary for protein-protein interactions, and these adducts contribute to interferences in its ability to facilitate apoptosome formation. To further characterize the structural and functional impact of these BQ-mediated PTMs, the original mixture of BQ-adducted cytochrome c was fractionated by liquid isoelectric focusing to provide various fractions of BQ-adducted cytochrome c species devoid of the native protein. The fractionation process separates samples based on their isoelectric point (pI), and because BQ adducts form predominantly on lysine residues, increased numbers of BQ adducts on cytochrome c correlate with a lower protein pI. Each fraction was analyzed for structural changes, and each was also assayed for the ability to support apoptosome-mediated activation of caspase-3. Circular dichroism revealed that several of the BQ-adducted cytochrome c species maintained a slightly more rigid structure in comparison to native cytochrome c. BQ-adducted cytochrome c also failed to activate caspase-3, with increasing numbers of BQ-lysine adducts corresponding to a greater inability to activate the apoptosome. In summary, the specific site of the BQ-lysine adducts, and the nature of the adduct, are important determinants of the subsequent structural changes to cytochrome c. In particular, adducts at sites necessary for protein-protein interactions interfere with the proapoptotic function of cytochrome c.

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. Quinones are a well-known class of electrophilic species. Many natural products contain quinones as active constituents, and the quinone moiety exists in a number of chemotherapeutic agents. Quinones are also frequently formed as electrophilic metabolites from a variety of xeno- and endobiotics. Hydroquinone (HQ) is present in the environment from various sources, and it is also a known metabolite of benzene. HQ is converted in the body to 1,4-benzoquinone, which subsequently gives rise to hematotoxic and nephrotoxic quinone-thioether metabolites. The toxicity of these metabolites is dependent upon their ability to arylate proteins and to produce oxidative stress. Protein tertiary structure and protein amino acid sequence combine to determine which proteins are targets of these electrophilic quinone-thioether metabolites. We have used cytochrome c and model peptides to view adduction profiles of quinone-thioether metabolites, and have determined by MALDI-TOF analysis that these electrophiles target specific residues within these model systems.

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. Recent advancements in mass spectrometry techniques and data analysis has permitted a more detailed investigation of site-specific protein modifications by reactive electrophiles. Knowledge from such analyses will assist in providing a better understanding of how specific classes of electrophiles produce toxicity and disease progression via site-selective protein-specific covalent modification. Hydroquinone (HQ) is a known environmental toxicant, and its quinone-thioether metabolites, formed via the intermediate generation of 1,4-benzoquinone (1,4-BQ), elicit their toxic response via the covalent modification of target proteins and the generation of reactive oxygen species. We have utilized a model protein, cytochrome c, to guide us in identifying 1,4-BQ- and 1,4-BQ-thioether derived site-specific protein modifications. LC-MS/MS analyses reveals that these modifications occur selectively on lysine and glutamic acid residues of the target protein, and that these modifications occur within identifiable "electrophile binding motifs" within the protein. These motifs are found within lysine-rich regions of the protein and appear to be target sites of 1,4-BQ-thioether adduction. These residues also appear to dictate the nature of post-adduction chemistry and the final structure of the adduct. This model system will provide critical insight for in vivo adduct hunting following exposure to 1,4-BQ-thioethers, but the general approaches can also be extended to the identification of protein adducts derived from other classes of reactive electrophiles.

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., 2 h) to Long Evans (wild-type Eker) rats, Western analysis utilizing an antibody specific for quinol-thioether metabolites of HQ revealed the presence of large amounts of chemical-protein adducts in both the OSOM and urine. By aligning the Western blot film with a parallel gel stained for protein, we can isolate the adducted proteins for LC-MS/MS analysis. Subsequent database searching can identify the specific site(s) of chemical adduction within these proteins. Finally, a combination of software programs can validate the identity of the adducted peptides. The site-specific identification of covalently adducted and oxidized proteins is a prerequisite for understanding the biological significance of chemical-induced posttranslational modifications (PTMs) and their toxicological significance.

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. MuDPIT was first described by Link et al. as a new technique useful for protein identification from a complex mixture of proteins (Link et al., Nat Biotechnol 17:676-682, 1999). MuDPIT utilizes two different HPLC columns to further enhance peptide separation, increasing the number of peptide hits and protein coverage. The technology is extremely useful for proteomes, such as the urine proteome, samples from immunoprecipitations, and 1D gel bands resolved from a tissue homogenate or lysate. In particular, MuDPIT has enhanced the field of adduct hunting for adducted peptides, since it is more capable of identifying lesser abundant peptides, such as those that are adducted, than the more standard LC-MS/MS. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response they elicit.

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. In this study, we have identified a number of proteins that are modified by quinone-thioethers after administration of 2-(glutathion-S-yl)HQ. Parallel one-dimensional gel electrophoresis was performed, and the Coomassie-stained gel was aligned with the corresponding Western blot, which was probed for adductions. Immunopositive bands were then subjected to trypsin digestion and analyzed via liquid chromatography/tandem mass spectrometry. The proteins that were subsequently identified contained a higher than average (9.7 versus 5.5%) lysine content and numerous stretches of lysine run-ons, which is a presumed electrophile binding motif. Approximately 50% of these proteins have also been identified as targets for electrophilic adduction by a diverse group of chemicals by other investigators, implying overlapping electrophile adductomes. By identifying a motif targeted by electrophiles it becomes possible to make predictions of proteins that may be targeted for adduction and possible sites on these proteins that are adducted. An understanding of proteins targeted for adduction is essential to unraveling the toxicity produced by these electrophiles.

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. Hydroquinone, and its mercapturic acid pathway metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of HQ and its thioether metabolites are in part a consequence of their oxidation to the corresponding electrophilic 1,4-benzoquinones (BQ). We now report that BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c. Furthermore, we have identified specific glutamic acid residues within cytochrome c as novel sites of NAC-BQ adduction. The microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. The solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Postadduction chemistry subsequently alters the nature of the final adduct. Using molecular modeling, the impact of BQ and NAC-BQ adduction on cytochrome c was visualized, revealing the spatial rearrangement of critical residues necessary for protein-protein interactions. Consequently, BQ-adducted cytochrome c fails to initiate caspase-3 activation in native lysates and also inhibits Apaf-1 oligomerization into an apoptosome complex in a purely reconstituted system. In summary, a combination of mass spectroscopic, molecular modeling, and biochemical approaches confirms that electrophile-protein adducts produce structural alterations that influence biological function.