Proteome Profiling of Rat Brain Cortical Changes during Early Postnatal Brain Development.

Label-free quantitation (LFQ) was applied to proteome profiling of rat brain cortical development during the early postnatal period. Male and female rat brain extracts were prepared using a convenient, detergent-free sample preparation technique at postnatal days (PND) 2, 8, 15, and 22. The PND protein ratios were calculated using Proteome Discoverer, and the PND protein change profiles were constructed separately for male and female animals for key presynaptic, postsynaptic, and adhesion brain proteins. The profiles were compared to the analogous profiles assembled from the published mouse and rat cortex proteomic data, including the fractionated-synaptosome data. The PND protein-change trendlines, Pearson correlation coefficient (PCC), and linear regression analysis of the statistically significant PND protein changes were used in the comparative analysis of the datasets. The analysis identified similarities and differences between the datasets. Importantly, there were significant similarities in the comparison of the rat cortex PND (current work) vs mouse (previously published) PND profiles, although in general, a lower abundance of synaptic proteins in mice than in rats was found. The male and female rat cortex PND profiles were expectedly almost identical (98-99% correlation by PCC), which also substantiated this LFQ nanoflow liquid chromatography-high-resolution mass spectrometry approach.

Developmental exposure to a commercial PBDE mixture: effects on protein networks in the cerebellum and hippocampus of rats.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are structurally similar to polychlorinated biphenyls (PCBs) and have both central (learning and memory deficits) and peripheral (motor dysfunction) neurotoxic effects at concentrations/doses similar to those of PCBs. The cellular and molecular mechanisms for these neurotoxic effects are not fully understood; however, several studies have shown that PBDEs affect thyroid hormones, cause oxidative stress, and disrupt Ca2+-mediated signal transduction. Changes in these signal transduction pathways can lead to differential gene regulation with subsequent changes in protein expression, which can affect the development and function of the nervous system. OBJECTIVE: In this study, we examined the protein expression profiles in the rat cerebellum and hippocampus following developmental exposure to a commercial PBDE mixture, DE-71. METHODS: Pregnant Long-Evans rats were dosed perinatally with 0 or 30.6 mg/kg/day of DE-71 from gestation day 6 through sampling on postnatal day 14. Proteins from the cerebellum and hippocampus were extracted, expression differences were detected by two-dimensional difference gel electrophoresis, and proteins were identified by tandem mass spectrometry. Protein network interaction analysis was performed using Ingenuity® Pathway Analysis, and the proteins of interest were validated by Western blotting. RESULTS: Four proteins were significantly differentially expressed in the cerebellum following DE-71 exposure, whereas 70 proteins were significantly differentially expressed in the hippocampus. Of these proteins, 4 from the cerebellum and 47 from the hippocampus, identifiable by mass spectrometry, were found to have roles in mitochondrial energy metabolism, oxidative stress, apoptosis, calcium signaling, and growth of the nervous system. CONCLUSIONS: Results suggest that changes in energy metabolism and processes related to neuroplasticity and growth may be involved in the developmental neurotoxicity of PBDEs.

Systematic proteomic approach to characterize the impacts of chemical interactions on protein and cytotoxicity responses to metal mixture exposures.

Chemical interactions have posed a big challenge in toxicity characterization and human health risk assessment of environmental mixtures. To characterize the impacts of chemical interactions on protein and cytotoxicity responses to environmental mixtures, we established a systems biology approach integrating proteomics, bioinformatics, statistics, and computational toxicology to measure expression or phosphorylation levels of 21 critical toxicity pathway regulators and 445 downstream proteins in human BEAS-2B cells treated with 4 concentrations of nickel, 2 concentrations each of cadmium and chromium, as well as 12 defined binary and 8 defined ternary mixtures of these metals in vitro. Multivariate statistical analysis and mathematical modeling of the metal-mediated proteomic response patterns showed a high correlation between changes in protein expression or phosphorylation and cellular toxic responses to both individual metals and metal mixtures. Of the identified correlated proteins, only a small set of proteins including HIF-1α is likely to be responsible for selective cytotoxic responses to different metals and metals mixtures. Furthermore, support vector machine learning was utilized to computationally predict protein responses to uncharacterized metal mixtures using experimentally generated protein response profiles corresponding to known metal mixtures. This study provides a novel proteomic approach for characterization and prediction of toxicities of metal and other chemical mixtures.

Novel molecular events associated with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat.

Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and testis (intact) proteomes in rats after 3 days of exposure. The adrenal accounted for most of the serum progesterone and all of the corticosterone increases in intact and castrated males. Serum luteinizing hormone, androstenedione, and testosterone in intact males shared a non-monotonic response suggesting transition from an acute stimulatory to a latent inhibitory response to exposure. Eight adrenal proteins were significantly altered with dose. There were unique proteomic changes between the adrenals of intact and castrated males. Six testis proteins in intact males had non-monotonic responses that significantly correlated with serum testosterone. Different dose-response curves for steroids and proteins in the adrenal and testis reveal novel adverse outcome pathways in intact and castrated male rats.

Proteomic analysis of mice expressing human ApoE demonstrates no differences in global protein solubility between APOE 3 and APOE 4 young mice.

Apolipoprotein E (ApoE) is a major lipid carrier protein. In humans, ApoE is expressed in three polymorphic isoforms, which are encoded by three different alleles APOE2, APOE3, and APOE4. In the brains of Alzheimer's disease (AD) patients, each one of these three allelic isoforms is found in several "isoelectric" protein isoforms (qPI), i.e. protein isoforms resulting from PTMs altering the net charge (q) of the polypeptide. AD is a complex disease in which multiple causes and several risk factors affect the onset and disease outcome. A major risk factor for AD is ApoE4; therefore, it is important to characterize the different ApoE qPIs. We have implemented a detergent-based method for isolation and quantitation of protein isoforms, and we found differences in the solubility of protein isoforms depending on the type of solvent used. In this manuscript, we describe these methods and applied them to young human-ApoE targeted replacement mice. Our results indicate that there are no significant differences in the hippocampus proteome of these mice as a function of the APOE genotype.

Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol.

Significant research has been focused on phthalate-induced alterations in male reproductive development. Studies on rodents have prompted the notion that a syndrome exists in the human male which includes phenotypic alterations such as hypospadias, cryptorchidism, poor semen quality, and even testicular cancer. Each phenotype in this 'testicular dysgenesis syndrome' is predicated on reduction in testosterone production by the fetal Leydig cell. We sought to examine the relationship between dysgenesis and steroidogenic capacity in the fetal rat testis more stringently by incorporating lower exposures than those typically used, conducting a comprehensive, non-targeted quantitative evaluation of the fetal testis proteome, and relating alterations in individual proteins to the capacity of the fetal Leydig cell to produce testosterone, and histopathology of the fetal testis. Pregnant dams were dosed orally from gestation day (GD) 13-19 with 0, 10, or 100 mg diethylhexyl phthalate (DEHP)/kg body weight per day. Each endpoint was represented by 16l. Clustering of Leydig cells occurred before any significant decrease in the capacity of the GD19 Leydig cell to produce testosterone. At 100 mg DEHP/kg, testosterone production was reduced significantly, Leydig cell clusters became quite large, and additional dysgenetic changes were observed in the fetal testis. Of 23 proteins whose expression was altered significantly at both DEHP exposure levels, seven were found to be correlated with and predictive of the quantified endpoints. None of these proteins have been previously implicated with DEHP exposure. Notably, pathway analysis revealed that these seven proteins fit a pathway network in which each is regulated directly or indirectly by estradiol.

Analysis of proteins using DIGE and MALDI mass spectrometry.

Difference gel electrophoresis (DIGE) is a common technique for characterizing differential protein expression in quantitative proteomics. Usually a combination of enzymatic digestion and peptide analysis by mass spectrometry is used to identify differentially expressed proteins following separation and statistical analysis by DIGE. In this chapter, methods for gel spot picking, enzymatic digestion, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for protein identification of DIGE-analyzed proteins are discussed. Two examples are given: first, a specific protein is used to test the sensitivity of the 2D DIGE/MALDI MS combination for protein quantification and identification, and second, several proteins with and without the labels typically used in DIGE are identified to demonstrate that these labels do not alter MS-based protein identification. Technical variations of protein gel spot preparation, in-gel digestion, and mass spectral protein identification are discussed.

Acute phase response, inflammation and metabolic syndrome biomarkers of Libby asbestos exposure.

Identification of biomarkers assists in the diagnosis of disease and the assessment of health risks from environmental exposures. We hypothesized that rats exposed to Libby amphibole (LA) would present with a unique serum proteomic profile which could help elucidate epidemiologically-relevant biomarkers. In four experiments spanning varied protocols and temporality, healthy (Wistar Kyoto, WKY; and F344) and cardiovascular compromised (CVD) rat models (spontaneously hypertensive, SH; and SH heart failure, SHHF) were intratracheally instilled with saline (control) or LA. Serum biomarkers of cancer, inflammation, metabolic syndrome (MetS), and the acute phase response (APR) were analyzed. All rat strains exhibited acute increases in α-2-macroglobulin, and α1-acid glycoprotein. Among markers of inflammation, lipocalin-2 was induced in WKY, SH and SHHF and osteopontin only in WKY after LA exposure. While rat strain- and age-related changes were apparent in MetS biomarkers, no LA effects were evident. The cancer marker mesothelin was increased only slightly at 1 month in WKY in one of the studies. Quantitative Intact Proteomic profiling of WKY serum at 1 day or 4 weeks after 4 weekly LA instillations indicated no oxidative protein modifications, however APR proteins were significantly increased. Those included serine protease inhibitor, apolipoprotein E, α-2-HS-glycoprotein, t-kininogen 1 and 2, ceruloplasmin, vitamin D binding protein, serum amyloid P, and more 1 day after last LA exposure. All changes were reversible after a short recovery regardless of the acute or long-term exposures. Thus, LA exposure induces an APR and systemic inflammatory biomarkers that could have implications in systemic and pulmonary disease in individuals exposed to LA.

Simultaneous detection of changes in protein expression and oxidative modification as a function of age and APOE genotype.

To better elucidate temporal changes in protein oxidation resulting from aging and the Alzheimer's disease-associated Apolipoprotein E (ApoE), we developed a 2D-DIGE-based method for simultaneously detecting differential expression and carbonyl oxidation of proteins. Specifically, we examined changes in the levels of oxidation and total protein expression in hippocampi from young-adult (25-30 weeks) and old (76-97 weeks) mice transgenic for the human Apolipoprotein E gene (APOE, APOE3, APOE4) isoforms, APOE3 or APOE4. Protein samples were labeled with either a fluorescent aminooxyacetamide (Alexa Fluor 488) to detect carbonyl modifications or with NHS-Cy3 to detect total protein expression. A protein sample used as an internal control was labeled with NHS-Cy5 and run on each gel. DIGE analysis revealed 38 differentially oxidized and 100 differentially expressed protein spots with significantly different levels (P < 0.05). For oxidized proteins, principal component analysis revealed two distinct clusters: one in which oxidation increased with age independent of APOE genotype, and the second in which oxidation was dependent on APOE genotype. For total protein expression, principal component analysis revealed a large overlap between changes with overall aging and between APOE genotypes. The use of a fluorescent tag to label oxidized proteins, in combination with a NHS-Cy3 to label total protein, makes it possible to determine changes in both protein oxidation and protein expression levels in a single experiment. These studies reveal that the expression levels of peroxiredoxin protein family members Prdx2, 3, and 6 are modified by age, APOE genotype, or both.

Early alterations in protein and gene expression in rat kidney following bromate exposure.

Bromate, a common disinfectant byproduct of drinking water ozonation, has been linked to human and animal renal toxicity, including renal cell carcinomas in multiple animal species. Here, we evaluate changes in protein and gene expression through two-dimensional difference gel electrophoresis (2D-DIGE) and Affymetrix arrays to identify potential modes of action involved in potassium bromate carcinogenicity. Male rats were exposed to potassium bromate in drinking water at concentrations of 0, 1, 20 and 400 ppm for two weeks. Differential expression of glycolytic proteins including enolase 1 (Eno1), triosephosphate isomerase 1 (Tpi1) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) suggests that bromate toxicity is associated with changes in energy consumption and utilization in renal cells involving up-regulation of glycolytic processes that may be the result of altered mitochondrial function. Several alterations in glycolysis and mitochondrial gene transcripts were also observed to be consistent with this mode of action. These studies provide insight into early events in renal cell physiology altered by bromate exposure.

Proteomic analysis optimization: selective protein sample on-column retention in reverse-phase liquid chromatography.

In an effort to optimize reverse-phase liquid chromatography (RPLC) for proteomics, we studied the impact of composition of the sample injection solution on protein on-column selection and retention. All the proteins studied were retained on-column when injections were made in 50% formic acid, 0.1% TFA or 8.3M urea. When formic acid was increased to 80%, the superoxide dismutase standard (MW 26,159) and 58 mouse microsomal proteins that possessed low-range molecular weights, high pIs or basic amino acid clusters were non-retained, resulting in retention selectivity during sample injection. Introducing to the 80% formic acid injection solution an organic solvent such as acetonitrile or acetonitrile-DMSO induced further retention selectivity, and increasing levels of organic solvents reduced on-column retention. The proteome was split into the proteins that were retained on-column which eluted at higher retention times (RTs), vs the proteins that collected in the injection flow-through which normally eluted at lower RTs. This protein selectivity was confirmed after fraction collection, 1D-GE and nano-LC-MS/MS. The significance of this procedure is that it can be exploited for fast extraction of small basic proteins from the bulk of the proteome and for on-column enrichment of hydrophobic proteins.

Measurement of oxidative stress parameters using liquid chromatography-tandem mass spectroscopy (LC-MS/MS).

There is increasingly intense scientific and clinical interest in oxidative stress and the many parameters used to quantify the degree of oxidative stress. However, there remain many analytical limitations to currently available assays for oxidative stress markers. Recent improvements in software, hardware, and instrumentation design have made liquid chromatography and tandem mass spectroscopy (LC-MS/MS) methods optimal choices for the determination of many oxidative stress markers. In particular, LC-MS/MS often provides the advantages of higher specificity, higher sensitivity, and the capacity to determine multiple analytes (e.g. 4-11 oxidative stress markers per LC run) when compared to other available methods, such as gas chromatography-MS, immunoassays, spectrophotometric or fluorometric assays. LC-MS/MS methods are also compatible with cleanup and sample preparation methods including prior solid phase extraction or automated two dimensional LC/LC chromatography followed by MS/MS. LC-MS/MS provides three analytical filtering functions: (1) the LC column provides initial separation as each analyte elutes from the column. (2) The first MS dimension isolates ions of a particular mass-to-charge (m/z) ratio. (3) The selected precursor ion is fragmented into product ions that provide structural information about the precursor ion. Quantitation is achieved based on the abundances of the product ions. The sensitivity limits for LC-MS/MS usually lie within the range of fg-pg of analyte per LC on-column injection. In this article, the present capabilities of LC-MS/MS are briefly presented and some specific examples of the strengths of these LC-MS/MS assays are discussed. The selected examples include methods for isoprostanes, oxidized proteins and amino acids, and DNA biomarkers of oxidative stress.

Continuous pH/salt gradient and peptide score for strong cation exchange chromatography in 2D-nano-LC/MS/MS peptide identification for proteomics.

Tryptic digests of human serum albumin and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MS/MS with strong cation exchange (SCX) and reversed-phase chromatography and continuous gradient elution. The peptide elution conditions combined simultaneous pH gradient with ammonium acetate salt gradient elution modes. A novel empirical SCX peptide elution score was developed, which accounts for both the number of basic and acidic residues and, in part, their location within a sequence of a peptide. Average scores calculated for the fractionated peptide sequences correlated well with the pH of SCX elution fractions. Multiple peptides with identical amino acid sequences, but differing in cysteine tags possessing different positive charge and different SCX elution properties, were obtained by subjecting the samples to reduction and alkylation with different cysteine alkylating reagents: iodoacetamide, 4-vinylpyridine, and (3-acrylamidopropyl) trimethylammonium chloride. The structurally similar peptides were used as elution standards.

Investigation of an enhanced resolution triple quadrupole mass spectrometer for high-throughput liquid chromatography/tandem mass spectrometry assays.

Triple quadrupole mass spectrometers, when operated in multiple reaction monitoring (MRM) mode, offer a unique combination of sensitivity, specificity, and dynamic range. Consequently, the triple quadrupole is the workhorse for high-throughput quantitation within the pharmaceutical industry. However, in the past, the unit mass resolution of quadrupole instruments has been a limitation when interference from matrix or metabolites cannot be eliminated. With recent advances in instrument design, triple quadrupole instruments now afford mass resolution of less than 0.1 Dalton (Da) full width at half maximum (FWHM). This paper describes the evaluation of an enhanced resolution triple quadrupole mass spectrometer for high-throughput bioanalysis with emphasis on comparison of selectivity, sensitivity, dynamic range, precision, accuracy, and stability under both unit mass (1 Da FWHM) and enhanced (