Multiomic Analyses of Dopaminergic Neurons Isolated from Human Substantia Nigra in Parkinson's Disease: A Descriptive and Exploratory Study.

Dopaminergic neurons (DA) of the substantia nigra pars compacta (SNpc) selectively and progressively degenerate in Parkinson's disease (PD). Until now, molecular analyses of DA in PD have been limited to genomic or transcriptomic approaches, whereas, to the best of our knowledge, no proteomic or combined multiomic study examining the protein profile of these neurons is currently available. In this exploratory study, we used laser capture microdissection to extract regions from DA in 10 human SNpc obtained at autopsy in PD patients and control subjects. Extracted RNA and proteins were identified by RNA sequencing and nanoliquid chromatography-mass spectrometry, respectively, and the differential expression between PD and control group was assessed. Qualitative analyses confirmed that the microdissection protocol preserves the integrity of our samples and offers access to specific molecular pathways. This multiomic analysis highlighted differential expression of 52 genes and 33 proteins, including molecules of interest already known to be dysregulated in PD, such as LRP2, PNMT, CXCR4, MAOA and CBLN1 genes, or the Aldehyde dehydrogenase 1 protein. On the other hand, despite the same samples were used for both analyses, correlation between RNA and protein expression was low, as exemplified by the CST3 gene encoding for the cystatin C protein. This is the first exploratory study analyzing both gene and protein expression of laser-dissected neuronal parts from SNpc in PD. Data are available via ProteomeXchange with identifier PXD024748 and via GEO with identifier GSE 169755.

Protein pathway analysis to study development-dependent effects of acute and repeated trimethyltin (TMT) treatments in 3D rat brain cell cultures.

Trimethyltin is an organometallic compound, described to be neurotoxic and to trigger neuroinflammation and oxidative stress. Previous studies associated TMT with the perturbation of mitochondrial function, or neurotransmission. However, the mechanisms of toxicity may differ depending on the duration of exposure and on the stage of maturation of brain cells. This study aim at elucidating whether the toxicity pathways triggered by a known neurotoxicant (TMT) differs depending on cell maturation stage or duration of exposure. To this end omics profiling of immature and differentiated 3D rat brain cell cultures exposed for 24 h or 10 days (10-d) to 0.5 and 1 µM of TMT was performed to better understand the underlying mechanisms of TMT associated toxicity. Proteomics identified 55 and 17 proteins affected by acute TMT treatment in immature and differentiated cultures respectively, while 10-day treatment altered 96 proteins in immature cultures versus 353 in differentiated. The results suggest different sensitivity to TMT depending on treatment duration and cell maturation. In accordance with known TMT mechanisms oxidative stress and neuroinflammation was observed after 10-d treatment at both maturation stages, whereas the neuroinflammatory process was more prominent in differentiated cultures than in the immature, no development-dependent difference could be detected for oxidative stress or synaptic neurodegeneration. Pathway analysis revealed that both vesicular trafficking and the synaptic machinery were strongly affected by 10-d TMT treatment in both maturation stages, as was GABAergic and glutamatergic neurotransmission. This study shows that omics approaches combined with pathway analysis constitutes an improved tool-set in elucidating toxicity mechanisms.

Detection of Unknown Chemical Adduct Modifications on Proteins: From Wet to Dry Laboratory.

The detection and characterization of chemical adducts on proteins is of increasing interest. Here, we described a step-by-step procedure to identify unknown chemical adduct modifications on proteins resulting from the interaction with a given reactive compound. The protocol can be divided into two equally important parts: (1) the wet laboratory work, to produce high quality mass spectrometry (MS) data of in vitro modified proteins and (2) the dry laboratory work, to analyze the generated MS data and provide highly confident qualitative and quantitative results on the chemical composition and amino acid localization of adducts. This protocol is applicable to the study of any pharmaceutical or chemical compound forming covalent protein adducts, detectable in LC-MS/MS experiments.

Detection of busulfan adducts on proteins.

RATIONALE: Busulfan is a bifunctional alkyl sulfonate antineoplastic drug. This alkylating agent was described as forming covalent adducts on proteins. However, only limited data are available regarding the interaction of busulfan with proteins. Mass spectrometry and bioinformatics were used to identify busulfan adducts on human serum albumin and hemoglobin. METHODS: Albumin and hemoglobin were incubated with busulfan or control compounds, digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Thermo Fisher LTQ Orbitrap Velos Pro. MS data were used to generate spectral libraries of non-modified peptides and an open modification search was performed to identify potential adduct mass shifts and possible modification sites. Results were confirmed by a second database search including identified mass shifts and by visual inspection of annotated tandem mass spectra of adduct-carrying peptides. RESULTS: Five structures of busulfan adducts were detected and a chemical structure could be attributed to four of them. Two were primary adducts corresponding to busulfan monoalkylation and alkylation of two amino acid residues by a single busulfan molecule. Two others corresponded to secondary adducts generated during sample processing. Adducts were mainly detected on Asp, Glu, and His residues. These findings were confirmed by subsequent database searches and experiments with synthetic peptides. CONCLUSIONS: The combination of in vitro incubation of proteins with the drug of interest or control compounds, high-resolution mass spectrometry, and open modification search allowed confirmation of the direct interaction of busulfan with proteins and characterization of the resulting adducts. Our results also showed that careful analysis of the data is required to detect experimental artifacts. Copyright © 2016 John Wiley & Sons, Ltd.

Repeated exposure to Ochratoxin A generates a neuroinflammatory response, characterized by neurodegenerative M1 microglial phenotype.

Neurotoxic effects of the environmentally abundant mycotoxin Ochratoxin A (OTA) were studied in histotypic 3D rat brain cell cultures, comprising all brain cell types. Cultures were exposed to nanomolar OTA concentrations and samples were collected 48h after a single exposure, or after 10 days of repeated administration. OTA-induced changes in gene- and protein expression, as well as alterations in cell morphology were assessed. Forty-eight-hour OTA exposure resulted in a disruption of the neuronal cytoskeleton and reduced expression of several oligodendrocyte-specific markers indicative of demyelination. Astrocyte disturbances were revealed by a decrease in two astrocytic proteins involved in regulation of inflammatory responses, metallothioneins I and II. Repeated OTA administration induced a neuroinflammatory response, as visualized by an increase of isolectin B4 labelled cells, increased expression of pro-inflammatory cytokines, and detection of macrophagic ED1/CD68 positive cells, as well as an upregulation of neurodegenerative M1 microglial phenotype markers. Partial recovery from OTA-induced deleterious effects on oligodendrocytes and astrocytes was achieved by co-treatment with sonic hedgehog (SHH). In addition, metallothionein I and II co-treatment partially restored OTA-induced effects on oligodendrocytes after 48h, and modulated microglial reactivity after 10 days. These results suggest that OTA-exposure affects Shh-signalling, which in turn may influence both oligodendrocytes and astrocytes. Furthermore, the primarily astrocytic proteins MTI/MTII may affect microglial activation. Thus the neuroinflammatory response appears to be downstream of OTA-induced effects on demyelination, axonal instabilities and astrocytes disturbances. In conclusion, repeated OTA-exposure induced a secondary neuroinflammatory response characterized by neurodegenerative M1 microglial activation and pro-inflammatory response that could exacerbate the neurodegenerative process.

Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures.

Xenobiotic exposure is a risk factor in the etiology of neurodegenerative disease. It was recently hypothesized that restricted exposure during brain development could predispose for a neurodegenerative disease later in life. As neuroinflammation contributes to progressive neurodegeneration, it is suspected that neurodevelopmental xenobiotic exposure could elicit a neuroinflammatory process, which over time may assume a detrimental character. We investigated the neurotoxic effects of paraquat (PQ) in three-dimensional whole rat brain cell cultures, exposed during an early differentiation stage, comparing immediate effects-directly post exposure-with long-term effects, 20 days after interrupted PQ-administration. Adverse effects and neuroinflammatory responses were assessed by measuring changes in gene- and protein-expression as well as by determining cell morphology changes. Differentiating neural cultures were highly susceptible to PQ and showed neuronal damage and strong astrogliosis. After the 20-day washout period, neurons partially recovered, whereas astrogliosis persisted, and was accompanied by microglial activation of a neurodegenerative phenotype. Our data shows that immediate and long-term effects of subchronic PQ-exposure differ. Also, PQ-exposure during this window of extensive neuronal differentiation led to a delayed microglial activation, of a character that could promote further pro-inflammatory signals that enable prolonged inflammation, thereby fueling further neurodegeneration.

Bile carcinoembryonic cell adhesion molecule 6 (CEAM6) as a biomarker of malignant biliary stenoses.

Differentiating malignant from nonmalignant biliary stenoses is challenging. This could be facilitated by the measurement of cancer biomarkers in bile. We aimed at (i) identifying new cancer biomarkers by comparative proteomic analysis of bile collected from patients with a malignant or benign biliary stenosis (exploratory phase) and (ii) verifying the accuracy of the newly identified potential biomarkers for discriminating malignant versus nonmalignant biliary stenoses in a larger group of patients (confirmation phase). Overall, 66 proteins were found overexpressed (ratio>1.5) in at least one cancer condition using proteomic analysis and 7 proteins were increased in all malignant/nonmalignant disease comparisons. Preliminary screening by immunoblot highlighted carcinoembryonic cell adhesion molecule 6 (CEAM6), a cell surface protein overexpressed in many human cancers, as an interesting candidate biomarker. ELISA subsequently confirmed CEAM6 as a potential bile biomarker for distinguishing malignant from benign biliary stenoses with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.92 (specificity 83%, sensitivity 93%, positive predictive value 93%, and negative predictive value 83%). No significant difference in serum CEAM6 level was found between malignant and nonmalignant samples. Combining bile CEAM6 and serum CA19-9 in a panel further improved diagnostic accuracy for malignant stenoses (AUC 0.96, specificity 83%, sensitivity 97%, positive predictive value 93%, and negative predictive value 91%). CEAM6 measurement in bile could be clinically useful to discriminate between malignant and nonmalignant causes of biliary stenosis. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Quantitative mass spectrometry analysis of intact hemoglobin A2 by precursor ion isolation and detection.

Precise and accurate quantification of proteins is essential in clinical laboratories. Here, we present a mass spectrometry (MS)-based method for the quantification of intact proteins in an ion trap mass spectrometer. The developed method is based on the isolation and detection of precursor ions for the quantification of the corresponding signals. The method was applied for the quantification of hemoglobin (Hb) A2, a marker used for the diagnosis of a ß-thalassemia trait. The α and δ globin chains, corresponding to total Hb and HbA2, respectively, were isolated in the ion trap at specific charge states and ejected without activation. Areas of the corresponding isolated precursor ions were used to calculate the δ to α ratio. Three series of quantifications were performed on 7 different days. The standard curve fitted linearly (R(2) = 0.9982) and allowed quantification of HbA2 over a concentration range from 3% to 18% of total Hb. Analytical imprecision ranged from 3.5% to 5.3%, which is enough to determine if the HbA2 level is below 3.5% or above 3.7%. In conclusion, our method reaches precision requirements that would be acceptable for the quantitative measurement of diagnostic proteins, such as HbA2, in clinical laboratories.

EasyProt--an easy-to-use graphical platform for proteomics data analysis.

High throughput protein identification and quantification analysis based on mass spectrometry are fundamental steps in most proteomics projects. Here, we present EasyProt (available at http://easyprot.unige.ch), a new platform for mass spectrometry data processing, protein identification, quantification and unexpected post-translational modification characterization. EasyProt provides a fully integrated graphical experience to perform a large part of the proteomic data analysis workflow. Our goal was to develop a software platform that would fulfill the needs of scientists in the field, while emphasizing ease-of-use for non-bioinformatician users. Protein identification is based on OLAV scoring schemes and protein quantification is implemented for both, isobaric labeling and label-free methods. Additional features are available, such as peak list processing, isotopic correction, spectra filtering, charge-state deconvolution and spectra merging. To illustrate the EasyProt platform, we present two identification and quantification workflows based on isobaric tagging and label-free methods.

Simultaneous quantification of ten cytotoxic drugs by a validated LC-ESI-MS/MS method.

A liquid chromatography separation with electrospray ionisation and tandem mass spectrometry detection method was developed for the simultaneous quantification of ten commonly handled cytotoxic drugs in a hospital pharmacy. These cytotoxic drugs are cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. The chromatographic separation was carried out by RPLC in less than 21 min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. MS/MS was performed on a triple quadrupole in selected reaction monitoring mode. The analytical method was validated to determine the limit of quantification (LOQ) and quantitative performance: lowest LOQs were between 0.25 and 2 ng mL(-1) for the ten investigated cytotoxic drugs; trueness values (i.e. recovery) were between 85% and 110%, and relative standard deviations for both repeatability and intermediate precision were always inferior to 15%. The multi-compound method was successfully applied for the quality control of pharmaceutical formulations and for analyses of spiked samples on potentially contaminated surfaces.