Tool For Proteomic Analysis Research Articles

Uromodulin and the study of urinary extracellular vesicles.

Urinary extracellular vesicles (uEVs) are a promising substrate for discovering new biomarkers. In order to investigate the origin of uEVs and the cargo they carry, some types of downstream analysis of uEVs may require concentration and enrichment as well as removal of contaminating substances. Co-isolation of the abundant urinary protein uromodulin with uEVs can be a problem, and may interfere with some techniques, in particular with proteomic analysis tools. Methods of separating out uromodulin and its removal have also not been standardized. This review highlights aspects of uromodulin structure that makes it recalcitrant to separation from uEVs, summarizes frequently used techniques for uEV enrichment and how they affect uromodulin separation, and specific methods for uromodulin removal during preparation of uEVs. The necessity of uromodulin removal for various study endpoints is also examined.

RECAPITULATING THE POST-SURGICAL BRAIN MICROENVIRONMENT OF ATYPICAL TERATOID RHABDOID TUMOURS TO IDENTIFY MEMBRANE PROTEINS FOR TARGETED THERAPY

Abstract AIMS Atypical Teratoid Rhabdoid Tumours (AT/RT) are rare aggressive tumours primarily arising in the hind brain of children under three, conferring median survival of <12 months. Surgery remains the only standard treatment; however tumours recur from post-surgical residual disease. Decellularised human brain cerebellum aim to recapitulate AT/RT post-surgical brain microenvironments which retain extracellular matrix (ECM) ligands, and where AT/RT plasma membrane proteins are hypothesised to represent viable molecular therapy targets. METHOD AT/RT, astrocyte and human brain cerebellum (decellularised and non-decellularised) membrane protein fractions were extracted and analysed using liquid chromatography-mass spectrometry. Proteomic analysis tools were integrated to identify differential expressed proteins and identify shared correlations. RESULTS Decellularised cerebellar ECM exhibited an enriched membrane proteome relative to total proteome, evidence of complete removal of intracellular components. Furthermore, decellularised and non-decellularised cerebellar tissue expressed comparable levels of membrane proteins and protein networks, indicative of ECM ligand retention. AT/RT and astrocyte KEGG Pathway analysis revealed specific oncology-based pathway expression in membrane protein compared to total protein, reinforcing membrane proteins as more visible therapeutic targets. Between the top 20 expressed proteins, ATP1A1 was the only membrane protein shared exclusively between all AT/RT cells and astrocytes, suggesting a critical functional role. Membrane proteins showing shared expression when normalised against each AT/RT cell line included ATP1A1, HSPD1, GNA13 and SLC3A2, indicating biological similarities between AT/RT molecular subtypes, which may offer candidate ubiquitous therapy targets. Membrane proteins expressed similarly in AT/RT-MYC molecular subtypes include ATP1A1, SLC7A5 and EEF1A1. STRING membrane pathway analysis also identified shared proteins between AT/RT and astrocyte cell populations. CONCLUSION AT/RT membrane protein pathways provide ideal therapeutic targets directly amenable for drug repurposing. Future work prioritises proteomic analysis utilising 3D AT/RT spheroids and cerebellum brain tissue to maximise accurately recapitulating the AT/RT post-surgical microenvironment. Cerebellum decellularisation still permits retention of essential proteins and ligands.

Whole genome sequencing of cr(VI) resistant Morganella morganii (1Ab1) towards identification of chromate transporters

Chromium (Cr) present naturally as the most abundant element on Earth’s crust. It usually exists combined with other chemical elements as oxygen and potassium in the form of chromium compounds. However, its level is raised due to leather tanning in tannery industries which turns less toxic trivalent chromium [Cr(III)] into highly toxic hexavalent chromium [Cr(VI)]. Chromium-resistant bacteria harbors Chr transporters in their genome which involves in metabolic reduction of toxic Cr to nontoxic Cr with the help of enzyme “chromate reductase.” This enzyme acts as a potential agent in bioremediation of highly chrome polluted sites. In this investigation, Whole Genome Sequencing (WGS) was done to find Chr transporters in the identified bacteria Morganella morganii (1Ab1) isolated from tannery effluent contaminated sites of Dindigul district, Tamil Nadu, India. Further, chr genes were confirmed using Real Time PCR and Web-based proteomic analysis tools such as Protter and Swiss Model. Finally output was reckoned for 3-D structure prediction and interactive visualization of the chr gene-encoded Chr protein that involves in Cr(VI) reduction.

Phage-Displayed SH2 Domain Libraries: From Ultrasensitive Tyrosine Phosphoproteome Probes to Translational Research.

Tyrosine phosphorylation is a critical regulator of cell signaling. A large fraction of the tyrosine phosphoproteome, however, remains uncharacterized, largely due to a lack of robust and scalable methods. The Src homology 2 (SH2) domain, a structurally conserved protein domain present in many intracellular signal-transducing proteins, naturally binds phosphorylated tyrosine (pTyr) residues, providing an ideal scaffold for the development of sensitive pTyr probes. Its modest affinity, however, has greatly limited its application. Phage display is an in vitro technique used for identifying ligands for proteins and other macromolecules. Using this technique, researchers have been able to engineer SH2 domains to increase their affinity and customize their specificity. Indeed, highly diverse phage display libraries have enabled the engineering of SH2 domains as affinity-purification (AP) tools for proteomic analysis as well as probes for aberrant tyrosine signaling detection and rewiring, and represent a promising class of novel diagnostics and therapeutics. This review describes the unique structure-function characteristics of SH2 domains, highlights the fundamental contribution of phage display in the development of technologies for the dissection of the tyrosine phosphoproteome, and highlights prospective uses of SH2 domains in basic and translational research.

Abstract 676: Neuro-oncology imaging mass cytometry panels enable spatial investigation of brain tumor microenvironment

Abstract Brain neoplasms represent a complex form of cancer that is one of the most challenging to classify and treat. Over 120 different tumor subtypes originate from various parts of the central nervous system, which makes identifying the composition of the tumor microenvironment (TME) vital for early assessment of progression, treatment, and prevention. We developed high-plex proteomic analysis tools to thoroughly characterize the TME of both human and mouse brain tissues using Imaging Mass Cytometry™ (IMC™). IMC offers unprecedented insight into the TME by uncovering the spatial distribution of 40-plus distinct molecular markers without autofluorescence, facilitating the research of brain neoplasms. Here, we demonstrate the application of high-plex human and mouse neuro-oncology IMC panels on normal and tumor formalin-fixed paraffin-embedded brain tissues. A basic neurophenotyping panel was developed and used to customize the Maxpar® Human and Maxpar OnDemand™ Mouse Immuno-Oncology IMC Panel Kits. Human and mouse neuro-oncology panels provide deep phenotyping and characterization of brain TME composition. These neuro panels consist of cross-reactive clones and enable flexible panel design for brain-specific research goals, such as brain tumor classification, and assessment of neuronal inflammation, degeneration, and development. We applied the neuro-oncology panels on tissue microarrays (TMAs) containing a variety of human brain tumors and mouse glioblastoma and neuroblastoma tissues. Normal brain tissues were used for comparative analysis as controls. The Hyperion™ Imaging System was utilized to digitize images from the tissues followed by quantitative analysis to assess the cellular composition of normal and cancerous brain TME. We successfully identified major cell populations that make up human and mouse brain matter, such as neurons, astrocytes, microglia, and oligodendrocytes. Various tumor cell phenotypes, resident and infiltrating cells, and resting and activated microglia were detected in multiple tumor subtypes. Subsequent single-cell analysis provided a comprehensive and quantitative assessment of the brain TME in our samples. We classified the distinct states of neurons and quantified myeloid and lymphoid immune cell infiltration across normal, astrocytoma, and glioblastoma tissues. Empowered by high-plex neuro-oncology panels, IMC can accelerate brain tumor research and provide insights into the spatial complexity of neuronal neoplasms. Citation Format: Nick Zabinyakov, Qanber Raza, Christina Loh. Neuro-oncology imaging mass cytometry panels enable spatial investigation of brain tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 676.

Proteomic Tools for the Analysis of Cytoskeleton Proteins.

Proteomic analyses have become an essential part of the toolkit of the molecular biologist, given the widespread availability of genomic data and open source or freely accessible bioinformatics software. Tools are available for detecting homologous sequences, recognizing functional domains, and modeling the three-dimensional structure for any given protein sequence, as well as for predicting interactions with other proteins or macromolecules. Although a wealth of structural and functional information is available for many cytoskeletal proteins, with representatives spanning all of the major subfamilies, the majority of cytoskeletal proteins remain partially or totally uncharacterized. Moreover, bioinformatics tools provide a means for studying the effects of synthetic mutations or naturally occurring variants of these cytoskeletal proteins. This chapter discusses various freely available proteomic analysis tools, with a focus on in silico prediction of protein structure and function. The selected tools are notable for providing an easily accessible interface for the novice while retaining advanced functionality for more experienced computational biologists.

Co-culturing multicellular tumor models: Modeling the tumor microenvironment and analysis techniques.

Advances in two-dimensional (2D) and three-dimensional (3D) cell culture over the last 10 years have led to the development of a plethora of methods for cultivating tumor models. More recently, cellular co-cultures have become a suitable testbed. The first portion of this review focuses on co-culturing methods that have been developed in recent years utilizing the multicellular tumor spheroid model. The latter portion describes techniques that are used to analyze the proteomes of mono- or co-cultured tumor models, with a focus on mass spectrometry (MS)-based analyses. Protein profiles are important indicators of the tumor heterogeneity. Therefore, there is a specific focus within this review on analysis by MS and MS imaging methods evaluating the proteomic profiles of 2D and 3D co-cultures. While these models are incredibly important for biological research, so far, they have not been widely explored on the proteomic level. With this review, we aim to introduce these systems to an analytical audience, with the goal of highlighting MS as an underutilized tool for proteomic analysis of tumor models.

The Glycoprotease CpaA Secreted by Medically Relevant Acinetobacter Species Targets Multiple O-Linked Host Glycoproteins.

Glycans decorate proteins and affect their biological function, including protection against proteolytic degradation. However, pathogenic, and commensal bacteria have evolved specific glycoproteases that overcome the steric impediment posed by carbohydrates, cleaving glycoproteins precisely at their glycosylation site(s). Medically relevant Acinetobacter strains employ their type II secretion system (T2SS) to secrete the glycoprotease CpaA, which contributes to virulence. Previously, CpaA was shown to cleave two O-linked glycoproteins, factors V and XII, leading to reduced blood coagulation. In this work, we show that CpaA cleaves a broader range of O-linked human glycoproteins, including several glycoproteins involved in complement activation, such as CD55 and CD46. However, only CD55 was removed from the cell surface, while CD46 remained unaltered during the Acinetobacter nosocomialis infection assay. We show that CpaA has a unique consensus target sequence that consists of a glycosylated serine or threonine residue after a proline residue (P-S/T), and its activity is not affected by sialic acids. Molecular modeling and mutagenesis analysis of CpaA suggest that the indole ring of Trp493 and the ring of the Pro residue in the substrate form a key interaction that contributes to CpaA sequence selectivity. Similar bacterial glycoproteases have recently gained attention as tools for proteomic analysis of human glycoproteins, and CpaA appears to be a robust and attractive new component of the glycoproteomics toolbox. Combined, our work provides insight into the function and possible application of CpaA, a member of a widespread class of broad-spectrum bacterial glycoproteases involved in host-pathogen interactions.IMPORTANCE CpaA is a glycoprotease expressed by members of the Acinetobacter baumannii-calcoaceticus complex, and it is the first bona fide secreted virulence factor identified in these species. Here, we show that CpaA cleaves multiple targets precisely at O-glycosylation sites preceded by a Pro residue. This feature, together with the observation that sialic acid does not impact CpaA activity, makes this enzyme an attractive tool for the analysis of O-linked human protein for biotechnical and diagnostic purposes. Previous work identified proteins involved in blood coagulation as targets of CpaA. Our work broadens the set of targets of CpaA, pointing toward additional roles in bacterium-host interactions. We propose that CpaA belongs to an expanding class of functionally defined glycoproteases that targets multiple O-linked host glycoproteins.

Joint pre-processing framework for two-dimensional gel electrophoresis images based on nonlinear filtering, background correction and normalization techniques

BackgroundTwo-dimensional gel electrophoresis (2-DGE) is a commonly used tool for proteomic analysis. This gel-based technique separates proteins in a sample according to their isoelectric point and molecular weight. 2-DGE images often present anomalies due to the acquisition process, such as: diffuse and overlapping spots, and background noise. This study proposes a joint pre-processing framework that combines the capabilities of nonlinear filtering, background correction and image normalization techniques for pre-processing 2-DGE images. Among the most important, joint nonlinear diffusion filtering, adaptive piecewise histogram equalization and multilevel thresholding were evaluated using both synthetic data and real 2-DGE images.ResultsAn improvement of up to 46% in spot detection efficiency was achieved for synthetic data using the proposed framework compared to implementing a single technique of either normalization, background correction or filtering. Additionally, the proposed framework increased the detection of low abundance spots by 20% for synthetic data compared to a normalization technique, and increased the background estimation by 67% compared to a background correction technique. In terms of real data, the joint pre-processing framework reduced the false positives up to 93%.ConclusionsThe proposed joint pre-processing framework outperforms results achieved with a single approach. The best structure was obtained with the ordered combination of adaptive piecewise histogram equalization for image normalization, geometric nonlinear diffusion (GNDF) for filtering, and multilevel thresholding for background correction.

Hyphenation of capillary zone electrophoresis with mass spectrometry for proteomic analysis: Optimization and comparison of two coupling interfaces

Capillary electrophoresis tandem mass spectrometry (CE–MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC–MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage.In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NH4Ac) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity.A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE–MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest.

IMOBILIZAÇÃO ENZIMÁTICA: CONCEITO E EFEITOS NA PROTEÓLISE

In recent years, great advances have been observed in the proteomic area regarding the different approaches adopted as top-down, middle-down and bottom-up. Regarding middle-down and bottom-up approaches, a previous step for protein digestion is necessary for further analysis of the generated peptides. Classical digestion protocols employing proteases are usually carried out in solution, which can lead to a number of disadvantages such as high digestion time, enzymatic autolysis and impossibility to reuse the enzymes. In view of these drawbacks, different approaches have been studied and applied for improving enzymatic efficiency, inhibition of autolysis, reduction of digestion time, and to minimize the number of missed cleavages. In the literature, great efforts have been made in order to achieve maximum efficiency in enzymatic digestion, with emphasis on proteases immobilization. In this review, concepts related to protein digestion, immobilization techniques, as well as the main support used for enzyme immobilization are discussed considering new tools for proteomic analysis.

Condiciones para el análisis de proteínas del micelio de Lentinula edodes obtenido por fermentación en estado líquido

Lentinula edodes es una seta comestible con potencial para el desarrollo de nutraceúticos. Sin embargo, son incipientes los trabajos enfocados en su producción biotecnológica y el desarrollo de herramientas analíticas que permitan profundizar en su composición. En esta investigación se estudió la producción de biomasa del hongo en el tiempo mediante fermentación en estado líquido y se seleccionaron las condiciones que permiten la obtención de extractos para la aplicación de herramientas para análisis proteómicos. Los métodos de extracción de proteínas, ácido tricloroacético (TCA)-Acetona y TCA-Acetona-Fenol, fueron comparados en términos del rendimiento de extracción y los perfiles de separación usando electroforesis en 1D (SDS-PAGE) y 2D (IEF-SDS PAGE). Se determinó que a los 10 días de crecimiento se obtiene la mayor producción de biomasa y proteína total. La extracción con TCA-Acetona-Fenol presentó un mayor rendimiento, mayor resolución y número de bandas en la electroforesis 1D. En 2DE los dos métodos permitieron la extracción de proteínas con puntos isoeléctricos en el rango de pH 3-10, pero el método TCA-Acetona-Fenol conllevó a una extracción diferencial, favoreciendo el rango de 33 a 113 kDa. Estos resultados se constituyen en una primera aplicación de técnicas de separación electroforética para futuros estudios proteómicos

HITS-PR-HHblits: protein remote homology detection by combining PageRank and Hyperlink-Induced Topic Search.

As one of the most important fundamental problems in protein sequence analysis, protein remote homology detection is critical for both theoretical research (protein structure and function studies) and real world applications (drug design). Although several computational predictors have been proposed, their detection performance is still limited. In this study, we treat protein remote homology detection as a document retrieval task, where the proteins are considered as documents and its aim is to find the highly related documents with the query documents in a database. A protein similarity network was constructed based on the true labels of proteins in the database, and the query proteins were then connected into the network based on the similarity scores calculated by three ranking methods, including PSI-BLAST, Hmmer and HHblits. The PageRank algorithm and Hyperlink-Induced Topic Search (HITS) algorithm were respectively performed on this network to move the homologous proteins of query proteins to the neighbors of the query proteins in the network. Finally, PageRank and HITS algorithms were combined, and a predictor called HITS-PR-HHblits was proposed to further improve the predictive performance. Tested on the SCOP and SCOPe benchmark datasets, the experimental results showed that the proposed protocols outperformed other state-of-the-art methods. For the convenience of the most experimental scientists, a web server for HITS-PR-HHblits was established at http://bioinformatics.hitsz.edu.cn/HITS-PR-HHblits, by which the users can easily get the results without the need to go through the mathematical details. The HITS-PR-HHblits predictor is a protocol for protein remote homology detection using different sets of programs, which will become a very useful computational tool for proteome analysis.

Qproteome FFPE Tissue Kit is not suitable for protein analysis using Agilent 2100 Bioanalyzer

Introduction: The chip-based protein separation using Agilent 2100 Bioanalyzer is a promising tool for proteomic analysis. However, it has not been defined if the above-mentioned device is suitable for analysis of formalin-fixed paraffin-embedded (FFPE) proteins extracts.<br>The aim of the study: Therefore we performed the analysis aimed at testing if extracts from FFPE tissues are suitable for proteins detection using Agilent 2100 Bioanalyzer.<br>Material and methods: FFPE tissues and cell cultures were used for experiments. Proteins were extracted from them using Qproteome FFPE Tissue Kit and RIPA buffer, respectively. For protein analysis Bioanalyzer Instrument, Western Blot and immunohistochemistry (IHC) were applied.<br>Results: In FFPE extracts, using Bioanalyzer we failed to detect β-actin. However, Western Blot analysis proved β-actin presence in them. One of possible explanations of this phenomenon might be lack of antibody-antigen interaction during immunoprecipitation preceding Bioanalyzer analysis. We suspected that, Extraction Buffer (from Qproteome FFPE Tissue Kit) or β-mercaptoethanol (both present in FFPE protein extracts) might be responsible for the above-mentioned blockade. Therefore, we applied IHC with detection of CD34 because, CD34 staining is robust against small methodological variations and this marker is always present in tumor tissue sections. Anti-CD34 antibody was diluted in Tris saline buffer with Extraction Buffer, β-mercaptoethanol or without. We did not observe CD34 immunopositivity only in the presence of Extraction Buffer.<br>Conclusion: Qproteome FFPE Tissue Kit is not suitable for protein analysis using Agilent 2100 Bioanalyzer, because the Extraction Buffer from the kit prevents an interaction between antibody and antigen during immunoprecipitation procedure.

New tool for proteomic analysis

New tool for proteomic analysis

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

Proteomic Tools for the Analysis of Cytoskeleton Proteins.

Proteomic analyses have become an essential part of the toolkit of the molecular biologist, given the widespread availability of genomic data and open source or freely accessible bioinformatics software. Tools are available for detecting homologous sequences, recognizing functional domains, and modeling the three-dimensional structure for any given protein sequence. Although a wealth of structural and functional information is available for a large number of cytoskeletal proteins, with representatives spanning all of the major subfamilies, the majority of cytoskeletal proteins remain partially or totally uncharacterized. Moreover, bioinformatics tools provide a means for studying the effects of synthetic mutations or naturally occurring variants of these cytoskeletal proteins. This chapter discusses various freely available proteomic analysis tools, with a focus on in silico prediction of protein structure and function. The selected tools are notable for providing an easily accessible interface for the novice, while retaining advanced functionality for more experienced computational biologists.

Transgenic tools for proteomic analysis of ciliary transport

Vision begins as photons are captured by photoreceptor cilia and light is converted into electrical signals that are then sent to the brain. As the photoreceptor cilium is not able to make its own proteins, all polypeptides needed for converting photons into electrical signals are synthetized in the cell body. How these molecules move from the cell body to cilia is still unclear. Opsin is one of the best-characterized transmembrane proteins. Our goal is to understand the mechanism of opsin transport into photoreceptor cilia. In this project, we use a combination of genetic and proteomic approaches in the zebrafish model. As the first step, we are constructing a transgenic line that expresses EGFP-opsin fusion from an inducible promoter specifically in photoreceptors. For this purpose, we will mate two transgenic lines: 1. A line that specifically expresses Cre recombinase in photoreceptors, and 2. A line that conditionally expresses EGFP-opsinCT44 (EGFP fused with the 44 C-terminal residues of opsin, which are sufficient to mediate ciliary transport) from the heat-shock promoter. A lox-mCherry-STOP cassette is inserted upstream of EGFP-opsinCT44. In the long run, we plan to purify photoreceptors via FACS sorting, pull down EGFP-opsinCT44 using the EGFP tag, and analyze opsin C-terminal binding proteins by mass spectrometry. In parallel, we will perform analysis of opsin transport in cilia mutants. We hope this will allow us to formulate a general model of how transmembrane proteins, such as GPCRs and TRP channels, are transported into cilia.

Assessing Hepatoxicants based on High-throughput Quantitative SILAC Proteomics and Causal Biological Networks

Drug-induced liver injury can result in the termination of compounds in preclinical development, as well as withdrawal of marketed drugs. Identification of the signaling pathways and proteins involved in injury is an important step towards establishing assays that could be utilized to identify potential hepatotoxicants early in the drug discovery process. In this study we used high throughput quantitative mass spectrometry proteomics involving Stable Isotope Labeling with Amino acids in Cell culture (SILAC) leveraged by a recently developed systems biology approach (Causal Reasoning Engine, CRE) to investigate the effects of hepatotoxic compounds on the cellular proteome of HepG2 cells. Cells were treated with various concentrations of nefazadone, nimesulide, nomifensine, or glafenine, all of which cause hepatotoxicity in humans. Buspirone and rosiglitazone were used as comparator compounds not associated with hepatotoxicity. In comparison to more traditional proteomic analysis tools CRE results provided detailed molecular hypotheses that condense into biological networks and collectively explain a significant number of the measured protein changes. The CRE hypotheses demonstrate that magnitude of response is not necessarily the differentiating factor between DILI and non-DILI compounds but rather the biological processes implicated. Differentiating CRE molecular hypotheses implicate lipid and glucose metabolism, inflammation, oxidative stress and DNA damage as consistent major in vitro discriminating factors. Overall, this paper provides evidence that SILAC data coupled with the CRE analysis method can provide context and new insight into variation of stress responses in hepatotoxic versus non-hepatotoxic compounds even within the same therapeutic class.