Method validation and new peak detection for the liquid chromatography-mass spectrometry multi-attribute method.

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry (LC-MS) peptide mapping technique that has been proposed as a replacement for several conventional quality control (QC) methods for therapeutic proteins. In addition to quantification of multiple product quality attributes (PQAs), MAM can also monitor impurities using a new peak detection (NPD) feature. Here, results are provided from method validation and NPD studies of an MAM approach applied to rituximab as a model monoclonal antibody (mAb). Twenty-one rituximab PQAs were monitored, including oxidation, pyroglutamination, deamidation, lysine clipping, and glycosylation. The PQA monitoring aspect of the method was validated according to ICH Guidance. Accuracy, precision, specificity, detection and quantitation limits, linearity, range, and robustness were demonstrated for this MAM approach with minimal issues. All PQAs were successfully validated except for several oxidation sites, which did not pass intermediate precision criteria. The variability found in oxidation measurements was attributed to artificial oxidation during sample preparation and could likely be alleviated through several approaches. The NPD aspect of the method was also evaluated. A spike-in approach was used to assess the limits of detection and quantitation (LOD/LOQ) of the NPD feature of MAM. For NPD, the peak intensity threshold was found to be the most critical parameter for accurate detection of impurities since a low threshold can result in false positives while a high threshold can obscure the detection of true peaks. Overall, the MAM approach presented and validated here has been demonstrated to be suitable for both targeted monitoring of rituximab PQAs and non-targeted detection of new peaks that represent impurities.

Correction to "Folic acid-conjugated liposomal vincristine for multidrug resistant cancer therapy".

[This corrects the article DOI: 10.1016/j.ajps.2013.07.015.].

Site-Specific Quantitation of Drug Conjugations on Antibody-Drug Conjugates (ADCs) Using a Protease-Assisted Drug Deconjugation and Linker-like Labeling (PADDLL) Method.

Antibody-drug conjugates (ADCs) are biopharmaceuticals for the targeted delivery of antitumor agents. ADCs can be highly heterogeneous with various drug-to-antibody ratio (DAR) species, conjugation sites, and occupancy levels. The conjugation site can modulate the ADC stability and efficacy and therefore can be considered to be a critical quality attribute (CQA) during development. Traditional mass spectrometry (MS)-based peptide mapping methods cannot accurately quantify site-specific conjugations due to a significant ionization discrepancy between unconjugated native peptides and conjugated peptides. Here, we developed a novel protease-assisted drug deconjugation and linker-like labeling (PADDLL) method to quantify the levels of linker payload at specific conjugation sites. We utilized optimized papain digestion to deconjugate the drug payload and labeled unoccupied conjugation sites with a linker-like structure to provide comparable ionization efficiency for MS-based quantitation. This method was successfully applied on two cysteine-linked, protease-cleavable ADCs, and the method demonstrated good linearity and reliability, reaching a limit of quantitation of below 1%. The calculated DARs were comparable with the results from intact mass analysis. The lot-to-lot variation in conjugation distribution and inferior conjugation stability at HC Cys225 to other interchain cysteines were observed. This method provides a valuable tool for ADC design and product development. To the best of our knowledge, this is the first analytical method developed to accurately quantify site-specific linker-drug payload conjugations for ADCs.

Long-chain fatty acyl-CoA synthetase 1 promotes prostate cancer progression by elevation of lipogenesis and fatty acid beta-oxidation.

Fatty acid metabolism is essential for the biogenesis of cellular components and ATP production to sustain proliferation of cancer cells. Long-chain fatty acyl-CoA synthetases (ACSLs), a group of rate-limiting enzymes in fatty acid metabolism, catalyze the bioconversion of exogenous or de novo synthesized fatty acids to their corresponding fatty acyl-CoAs. In this study, systematical analysis of ACSLs levels and the amount of fatty acyl-CoAs illustrated that ACSL1 were significantly associated with the levels of a broad spectrum of fatty acyl-CoAs, and were elevated in human prostate tumors. ACSL1 increased the biosynthesis of fatty acyl-CoAs including C16:0-, C18:0-, C18:1-, and C18:2-CoA, triglycerides and lipid accumulation in cancer cells. Mechanistically, ACSL1 modulated mitochondrial respiration, ß-oxidation, and ATP production through regulation of CPT1 activity. Knockdown of ACSL1 inhibited the cell cycle, and suppressed the proliferation and migration of prostate cancer cells in vitro, and growth of prostate xenograft tumors in vivo. Our study implicates ACSL1 as playing an important role in prostate tumor progression, and provides a therapeutic strategy of targeting fatty acid metabolism for the treatment of prostate cancer.

Identification of lysine isobutyrylation as a new histone modification mark.

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.

Long-Chain Acyl-CoA Synthetase 4-Mediated Fatty Acid Metabolism Sustains Androgen Receptor Pathway-Independent Prostate Cancer.

Androgen deprivation therapy has led to elevated cases of androgen receptor (AR) pathway-independent prostate cancer with dysregulated fatty acid metabolism. However, it is unclear how prostate cancer cells sustain dysregulated fatty acid metabolism to drive AR-independent prostate cancer. Long-chain acyl-CoA synthetases (ACSL) catalyze the conversion of fatty acids into fatty acyl-CoAs that are required for fatty acid metabolism. In this study, we demonstrate that expression levels of ACSL3 and 4 were oppositely regulated by androgen-AR signaling in prostate cancer cells. AR served as a transcription suppressor to bind at the ACSL4 promoter region and inhibited its transcription. Inhibition of androgen-AR signaling significantly downregulated ACSL3 and PSA, but elevated ACSL4 levels. ACSL4 regulated a broad spectrum of fatty acyl-CoA levels, and its catalytic efficiency in fatty acyl-CoAs biosynthesis was about 1.9- to 4.3-fold higher than ACSL3. In addition, in contrast to ACSL3, ACSL4 significantly regulated global protein myristoylation or myristoylation of Src kinase in prostate cancer cells. Knockdown of ACSL4 inhibited the proliferation, migration, invasion, and xenograft growth of AR-independent prostate cancer cells. Our results suggest that the surge of ACSL4 levels by targeting AR signaling increases fatty acyl-CoAs biosynthesis and protein myristoylation, indicating the opposite, yet complementary or Yin-Yang regulation of ACSL3 and 4 levels in sustaining fatty acid metabolism when targeting androgen-AR signaling. This study reveals a mechanistic understanding of ACSL4 as a potential therapeutic target for treatment of AR-independent prostate cancer. IMPLICATIONS: AR coordinately regulates the expression of ACSL3 and ACSL4, such that AR pathway-independent prostate tumors become dependent on ACSL4-mediated fatty acid metabolism.

Oral quetiapine treatment results in time-dependent alterations of recognition memory and brain-derived neurotrophic factor-related signaling molecules in the hippocampus of rats.

Antipsychotic drugs (APDs) have a variety of important therapeutic applications for neuropsychiatric disorders. However, they are routinely prescribed off-label across all age categories, a controversial practice given their potential for producing metabolic and extrapyramidal side effects. Evidence also suggests that chronic treatment with some APDs may lead to impairments in cognition and decreases in brain volume, although these findings are controversial. The purpose of the studies described here was to evaluate one of the most commonly prescribed APDs, quetiapine, for chronic effects on recognition memory, brain-derived neurotrophic factor (BDNF), its precursor proBDNF, as well as relevant downstream signaling molecules that are known to influence neuronal plasticity and cognition. Multiple cohorts of adult rats were treated with quetiapine (25.0 mg/kg/day) for 30 or 90 days in their drinking water then evaluated for drug effects on motor function in a catalepsy assessment, recognition memory in a spontaneous novel object recognition (NOR) task, and BDNF-related signaling molecules in the post mortem hippocampus via Western Blot. The results indicated that oral quetiapine at a dose that did not induce catalepsy, led to time-dependent impairments in NOR performance, increases in the proBDNF/BDNF ratio, and decreases in Akt and CREB phosphorylation in the hippocampus. These results indicate that chronic treatment with quetiapine has the potential to adversely affect recognition memory and neurotrophin-related signaling molecules that support synaptic plasticity and cognitive function. Given the widespread use this APD across multiple conditions and patient populations, such long-term effects observed in animals should be considered.

Chronic oral treatment with risperidone impairs recognition memory and alters brain-derived neurotrophic factor and related signaling molecules in rats.

Antipsychotic drugs (APDs) are essential for the treatment of schizophrenia and other neuropsychiatric illnesses such as bipolar disease. However, they are also extensively prescribed off-label for many other conditions, a practice that is controversial given their potential for long-term side effects. There is clinical and preclinical evidence that chronic treatment with some APDs may lead to impairments in cognition and decreases in brain volume, although the molecular mechanisms of these effects are unknown. The purpose of the rodent studies described here was to evaluate a commonly prescribed APD, risperidone, for chronic effects on recognition memory, brain-derived neurotrophic factor (BDNF), its precursor proBDNF, as well as relevant downstream signaling molecules that are known to influence neuronal plasticity and cognition. Multiple cohorts of adult rats were treated with risperidone (2.5 mg/kg/day) or vehicle (dilute acetic acid solution) in their drinking water for 30 or 90 days. Subjects were then evaluated for drug effects on recognition memory in a spontaneous novel object recognition task and protein levels of BDNF-related signaling molecules in the hippocampus and prefrontal cortex. The results indicated that depending on the treatment period, a therapeutically relevant daily dose of risperidone impaired recognition memory and increased the proBDNF/BDNF ratio in the hippocampus and prefrontal cortex. Risperidone treatment also led to a decrease in Akt and CREB phosphorylation in the prefrontal cortex. These results indicate that chronic treatment with a commonly prescribed APD, risperidone, has the potential to adversely affect recognition memory and neurotrophin-related signaling molecules that support synaptic plasticity and cognitive function.

Glycan analysis for protein therapeutics.

Glycosylation can be a critical quality attribute for protein therapeutics due to its extensive impact on product safety and efficacy. Glycan characterization is important in the process of protein drug development, from early stage candidate selection to late stage regulatory submission. It is also an indispensable part in the evaluation of biosimilarity. This review discusses the effects of glycosylation on the stability and activity of protein therapeutics, regulatory considerations corresponding to manufacturing and structural characterization of glycosylated protein therapeutics, and focuses on mass spectrometry compatible separation methods for glycan characterization of protein therapeutics. These approaches include hydrophilic interaction liquid chromatography, reversed-phase liquid chromatography, capillary electrophoresis, porous graphitic carbon liquid chromatography and two-dimensional liquid chromatography. Advances and novelties in each separation method, as well as associated challenges and limitations, are discussed at the released glycan, glycopeptide, glycoprotein subunit and intact glycoprotein levels.

Dietary palmitate cooperates with Src kinase to promote prostate tumor progression.

Numerous genetic alterations have been identified during prostate cancer progression. The influence of environmental factors, particularly the diet, on the acceleration of tumor progression is largely unknown. Expression levels and/or activity of Src kinase are highly elevated in numerous cancers including advanced stages of prostate cancer. In this study, we demonstrate that high-fat diets (HFDs) promoted pathological transformation mediated by the synergy of Src and androgen receptor in vivo. Additionally, a diet high in saturated fat significantly enhanced proliferation of Src-mediated xenograft tumors in comparison with a diet high in unsaturated fat. The saturated fatty acid palmitate, a major constituent in a HFD, significantly upregulated the biosynthesis of palmitoyl-CoA in cancer cells in vitro and in xenograft tumors in vivo. The exogenous palmitate enhanced Src-dependent mitochondrial ß-oxidation. Additionally, it elevated the amount of C16-ceramide and total saturated ceramides, increased the level of Src kinase localized in the cell membrane, and Src-mediated downstream signaling, such as the activation of mitogen-activated protein kinase and focal adhesion kinase. Our results uncover how the metabolism of dietary palmitate cooperates with elevated Src kinase in the acceleration of prostate tumor progression.

Mass Spectrometric Quantitation of Tubulin Acetylation from Pepsin-Digested Rat Brain Tissue Using a Novel Stable-Isotope Standard and Capture by Anti-Peptide Antibody (SISCAPA) Method.

Acetylation of α-tubulin at Lys-40 is a potential biomarker for cognitive deficits in various neurological disorders. However, this key post-translational modification (PTM) has not been previously studied with mass spectrometry, due to the inadequate distribution of tryptic cleavage sites. Following peptic digestion, a surrogate sequence containing this key PTM site was identified and was found to be stable and quantitatively reproducible. A highly sensitive and specific SISCAPA-LC-MS method for quantitating rat brain tubulin acetylation was developed, validated, and applied, and only required a small amount of tissue (2.2 mg). This workflow includes peptic digestion, stable-isotope dilution, capture with antiacetylated peptide antibody bound on protein G beads, and quantitation using LC-MS. The method allowed a lower limit of quantitation at 2.50 pmol/mg and provided a linear range of 2.50-62.50 pmol/mg. Selectivity, intra and interday precision and accuracy were also validated. This method has been successfully applied in a preclinical study of organophosphate neurotoxicity, and we found that chronic exposure to chlorpyrifos led to a significant and persistent inhibition of brain tubulin acetylation.

Revealing the protein propionylation activity of the histone acetyltransferase MOF (males absent on the first).

Short-chain acylation of lysine residues has recently emerged as a group of reversible posttranslational modifications in mammalian cells. The diversity of acylation further broadens the landscape and complexity of the proteome. Identification of regulatory enzymes and effector proteins for lysine acylation is critical to understand functions of these novel modifications at the molecular level. Here, we report that the MYST family of lysine acetyltransferases (KATs) possesses strong propionyltransferase activity both in vitro and in cellulo Particularly, the propionyltransferase activity of MOF, MOZ, and HBO1 is as strong as their acetyltransferase activity. Overexpression of MOF in human embryonic kidney 293T cells induced significantly increased propionylation in multiple histone and non-histone proteins, which shows that the function of MOF goes far beyond its canonical histone H4 lysine 16 acetylation. We also resolved the X-ray co-crystal structure of MOF bound with propionyl-coenzyme A, which provides a direct structural basis for the propionyltransferase activity of the MYST KATs. Our data together define a novel function for the MYST KATs as lysine propionyltransferases and suggest much broader physiological impacts for this family of enzymes.

Tropisetron enhances recognition memory in rats chronically treated with risperidone or quetiapine.

While impairments of cognition in schizophrenia have the greatest impact on long-term functional outcome, the currently prescribed treatments, antipsychotic drugs (APDs), do not effectively improve cognition. Moreover, while more than 20 years have been devoted to the development of new drugs to treat cognitive deficits in schizophrenia, none have been approved to date. One area that has not been given proper attention at the preclinical or clinical stage of drug development is the chronic medication history of the test subject. Hence, very little is known about how chronic treatment with drugs that affect multiple receptors like APDs influence the response to a potential pro-cognitive agent. Therefore, the purpose of this study was to evaluate the α7 nicotinic acetylcholine receptor (α7 nAChR) partial agonist, tropisetron in rats chronically treated with APDs with distinct pharmacological profiles. Rats were treated orally with either risperidone (2.5 mg/kg/day) or quetiapine (25.0 mg/kg/day) for 30 or 90 days and then an acute injection of vehicle or tropisetron (3.0 mg/kg) was administered before training in a novel object recognition (NOR) task. After a 48 h delay (when recollection of the familiar object was impaired in vehicle-treated animals) neither 30 nor 90 days of risperidone or quetiapine treatment improved NOR performance. In contrast, tropisetron markedly improved NOR performance in rats treated with either APD for 30 or 90 days. These animal data reinforce the argument that two commonly prescribed APDs are not pro-cognitive agents and that α7 nAChR ligands like tropisetron have potential as adjunctive treatments in schizophrenia.

Determination of chlorpyrifos and its metabolites in cells and culture media by liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive method to simultaneously quantitate chlorpyrifos, chlorpyrifos oxon and the detoxified product 3,5,6-trichloro-2-pyridinol (TCP) was developed using either liquid-liquid extraction for culture media samples, or protein precipitation for cell samples. Multiple reaction monitoring in positive ion mode was applied for the detection of chlorpyrifos and chlorpyrifos oxon, and selected ion recording in negative mode was applied to detect TCP. The method provided linear ranges from 5 to 500, 0.2-20 and 20-2000ng/mL for media samples and from 0.5-50, 0.02-2 and 2-200ng/million cells for CPF, CPO and TCP, respectively. The method was validated using selectivity, linearity, precision, accuracy, recovery, stability and dilution tests. All relative standard deviations (RSDs) and relative errors (REs) for QC samples were within 15% (except for LLOQ, within 20%). This method has been successfully applied to study the neurotoxicity and metabolism of chlorpyrifos in a human neuronal model.

Myristoylation of Src kinase mediates Src-induced and high-fat diet-accelerated prostate tumor progression in mice.

Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression.

Chlorpyrifos and chlorpyrifos oxon impair the transport of membrane bound organelles in rat cortical axons.

Chlorpyrifos (CPF) is an extensively used organophosphorus pesticide that has recently come under increasing scrutiny due to environmental health concerns particularly its association with neurodevelopmental defects. While the insecticidal actions and acute toxicity of CPF are attributed to its oxon metabolite (CPO) which potently inhibits the cholinergic enzyme acetylcholinesterase (AChE), there is significant evidence that CPF, CPO, and other organophosphates may affect a variety of neuronal targets and processes that are not directly related to AChE. Previously, in adult rat sciatic nerves ex vivo and postnatal neurons from rats in vitro we observed that CPF and CPO impaired the movements of vesicles and mitochondria in axons. Here, in embryonic neurons from rats in culture, we evaluated 24h exposures to CPF and CPO across picomolar to micromolar concentrations for effects on fast axonal transport of membrane bound organelles (MBOs) that contained the amyloid precursor protein (APP) tagged with the fluorescent marker, Dendra2 (APPDendra2). The most notable observations of this study were concentration-dependent decreases in the velocity and percentage of MBOs moving in the anterograde direction, an increase in the number of stationary MBOs, and an increased frequency of pauses associated with both CPF and CPO. These effects occurred at concentrations that did not significantly inhibit AChE activity, they were not blocked by cholinergic receptor antagonists, and they were not associated with compromised cell viability. These effects of CPF and CPO may be significant given the importance of axonal transport to neuronal development as well the function of fully developed neurons.

Profiling Cellular Substrates of Lysine Acetyltransferases GCN5 and p300 with Orthogonal Labeling and Click Chemistry.

p300 and GCN5 are two representative lysine acetyltransferases (KATs) in mammalian cells. It was recently reported that they possess multiple acyltransferase activities including acetylation, propionylation, and butyrylation of the ε-amino group of lysine residues of histones and non-histone protein substrates. Although thousands of acetylated substrates and acetylation sites have been identified by mass spectrometry-based proteomic screening, our knowledge about the causative connections between individual KAT members and their corresponding sub-acylomes remain very limited. Herein, we applied 3-azidopropionyl CoA (3AZ-CoA) as a bioorthogonal surrogate of acetyl-, propionyl- and butyryl-CoA for KAT substrate identification. We successfully attached the azide as a chemical warhead to cellular substrates of wild-type p300 and engineered GCN5. The substrates were subsequently labeled with biotin tag through the copper-catalyzed azide-alkyne cycloaddition (CuAAC). Following protein enrichment on streptavidin-coated resin, we conducted LC-MS/MS studies from which more than four hundred proteins were identified as GCN5 or p300 substrate candidates. These proteins are either p300- or GCN5-unique or shared by the two KATs and are extensively involved in various biological events including gene expression, cell cycle, and cellular metabolism. We also experimentally validated two novel substrates of GCN5, that is, IQGAP1 and SMC1. These results demonstrate extensive engagement of GCN5 and p300 in cellular pathways and provide new insights into understanding their functions in specific biological processes.

From the Cover: AstrocytesAre Protective Against Chlorpyrifos Developmental Neurotoxicity in Human Pluripotent Stem Cell-Derived Astrocyte-Neuron Cocultures.

Human neural progenitor cells are capable of independent, directed differentiation into astrocytes, oligodendrocytes and neurons and thus offer a potential cell source for developmental neurotoxicity (DNT) systems. Human neural progenitor-derived astrocyte-neuron cocultured at defined ratios mimic cellular heterogeneity and interaction in the central nervous system. Cytochrome P450 enzymes are expressed at a relatively high level in astrocytes and may play a critical role in the biotransformation of endogenous or exogenous compounds, including chlorpyrifos, an organophosphate insecticide that affects the central nervous system. P450 enzymes metabolize chlorpyrifos to chlorpyrifos-oxon, which is then metabolized primarily to 3, 5, 6-trichloropyridinol in addition to diethylphosphate and diethylthiophosphate. These end metabolites are less neurotoxic than chlorpyrifos and chlorpyrifos-oxon. Our objective was to identify the interactive role of astrocytes and neurons in chlorpyrifos-induced human DNT. In neuron-only cultures, chlorpyrifos inhibited neurite length, neurite number and branch points per neuron in a dose-dependent manner during a 48 h exposure, starting at 10 µM. However, in astrocyte-neuron cocultures, astrocytes protected neurons from the effects of chlorpyrifos at higher concentrations, up to and including 30 µM chlorpyrifos and endogenous astrocyte P450 enzymes effectively metabolized chlorpyrifos. The P450 inhibitor SKF525A partly negated the protective effect of astrocytes, allowing reduction in branch points with chlorpyrifos (10 µM). Thus, the scalable and defined astrocyte-neuron cocultures model that we established here has potentially identified a role for P450 enzymes in astrocytic neuroprotection against chlorpyrifos and provides a novel model for addressing DNT in a more accurate multicellular environment.

Development of a Method for the Determination of Acyl-CoA Compounds by Liquid Chromatography Mass Spectrometry to Probe the Metabolism of Fatty Acids.

Acyl-Coenzyme As (acyl-CoAs) are a group of activated fatty acid molecules participating in multiple cellular processes including lipid synthesis, oxidative metabolism of fatty acids to produce ATP, transcriptional regulation, and protein post-translational modification. Quantification of cellular acyl-CoAs is challenging due to their instability in aqueous solutions and lack of blank matrices. Here we demonstrate an LC-MS/MS analytical method which allows for absolute quantitation with broad coverage of cellular acyl-CoAs. This assay was applied to profile endogenous acyl-CoAs under the challenge of a variety of dietary fatty acids in prostate and hepatic cells. Additionally, this approach allowed for detection of multiple fatty acid metabolic processes including the biogenesis of acyl-CoAs, and their elongation, degradation, and desaturation. Hierarchical clustering in the remodeling of acyl-CoA profiles revealed a fatty-acid-specific pattern across all tested cell lines, which provides a valuable reference for making predictions in other cell models. Individual acyl-CoAs were identified which were altered differentially by exogenous fatty acids in divergent tumorigenicity states of cells. These findings demonstrate the power of acyl-CoA profiling toward understanding the mechanisms for the progression of tumors or other diseases in response to fatty acids.

Identification of protein adduction using mass spectrometry: Protein adducts as biomarkers and predictors of toxicity mechanisms.

The determination of protein-xenobiotic adducts using mass spectrometry is an emerging area which allows detailed understanding of the underlying mechanisms involved in toxicity. These approaches can also be used to reveal potential biomarkers of exposure or toxic response. The following review covers studies of protein adducts resulting from exposure to a wide variety of xenobiotics including organophosphates, polycyclic aromatic hydrocarbons, acetaminophen, alkylating agents and other related compounds.