Process-driven information management system at a biotech company: concept and implementation

While established pharmaceutical companies have chemical information systems in place to manage their compounds and the associated data, new startup companies need to implement these systems from scratch. Decisions made early in the design phase usually have long lasting effects on the expandability, maintenance effort, and costs associated with the information management system. Careful analysis of work and data flows, both inter- and intradepartmental, and identification of existing dependencies between activities are important. This knowledge is required to implement an information management system, which enables the research community to work efficiently by avoiding redundant registration and processing of data and by timely provision of the data whenever needed. This paper first presents the workflows existing at Anadys, then ARISE, the research information management system developed in-house at Anadys. ARISE was designed to support the preclinical drug discovery process and covers compound registration, analytical quality control, inventory management, high-throughput screening, lower throughput screening, and data reporting.     

一种规模化蛋白质组分离和鉴定新方法研究及其应用

建立了一种规模化的蛋白质组分离和鉴定新方法。通过对在生命发育过程中具有重要研究价值的人胎肝线粒体蛋白质组的分离分析，表明与毛细管液-质联用的不同分离方法的组合可以增大检测动态范围和分辨率。研究共鉴定了2977个肽段，归属于915种蛋白质。去除批次间冗余后，鉴定的蛋白质为477种，其中291种为唯一蛋白质，186种为蛋白质簇，144种蛋白质明确定位于人胎肝线粒体中。所鉴定蛋白质的分子量分布范围为7000Da～330000 Da，pI值分布在4．0～11．89，克服了两维凝胶电泳在分子量和pH方面的歧视性问题。实验中发现的蛋白质簇以及确定一种蛋白质需要最少肽段数的问题还需要进一步研究。     

Comparative Proteomics of Potato Cultivars with a Variable Dormancy Period

The control of the duration of the dormancy phase is a significant challenge in the potato industry and for seed producers. However, the proteome landscape involved in the regulation of the length of the dormancy period over potato cultivars remains largely unexplored. In this study, we performed for the first time a comparative proteome profiling of potato cultivars with differential duration of tuber dormancy. More specifically, the proteome profiling of Agata, Kennebec and Agria commercial potato varieties with short, medium and medium-long dormancy, respectively, was assessed at the endodormancy stage using high-resolution two-dimensional electrophoresis (2-DE) coupled to reversed-phase liquid chromatography–tandem mass spectrometry (LC-TripleTOF MS/MS). A total of 11 proteins/isoforms with statistically significant differential abundance among cultivars were detected on 2-DE gels and confidently identified by LC-TripleTOF MS/MS. Identified proteins have known functions related to tuber development, sprouting and the oxylipins biosynthesis pathway. Fructokinase, a mitochondrial ADP/ATP carrier, catalase isozyme 2 and heat shock 70 kDa were the proteins with the strongest response to dormancy variations. To the best of our knowledge, this study reports the first candidate proteins underlying variable dormancy length in potato cultivars.     

Proteomics of human cancer tissues and cells

Proteomic analysis of cancer tissues and cells provides valuable information to identify promising targets for cancer diagnosis, prognosis and therapy. Novel strategies have emerged to optimize the workflow of tissue procurement, and tissue and cell selection, and to improve protocols for the extraction of protein from fresh, frozen and paraffin-embedded tissue. Moreover, in the context of advanced approaches to proteomics, mass spectrometry and array-based technologies strongly contribute to protein profiling of cancer tissues and cells.The focus of this review is the methods by which all the steps of a proteomic investigation on human-cancer tissue (from choice of the experimental model to validation of candidate biomarkers) should be performed, paying particular attention to recently developed strategies. The review also presents an overview of the most recent high-throughput proteomic studies in cancer research.     

Proteomics of metal transport and metal-associated diseases

Proteomics technology has the potential to identify groups of proteins that have similar biological function. However, few attempts have been made to identify and characterize metal-binding proteins by using proteomics strategies. Many transition metals are essential to sustain life. Copper, iron, and zinc are the most abundant transition metals relevant to biological systems. In addition to their important biological functions, metals can also catalyze the formation of damaging free radical species. Hence, their intracellular transport is tightly regulated. Despite recent insights into the intracellular transport of copper and other metals, our overall understanding of intracellular metal metabolism remains incomplete and it is likely that many metal-binding proteins remain undiscovered. Furthermore, the protein targets for metals during metal-associated disease states or during exposure to toxic levels of environmental metals are yet to be unravelled. A proteomics strategy for the analysis of metal-transporting or metal-binding proteins has the potential to uncover how a large number of proteins function in normal or metal-associated diseased states. Here we discuss the principal aspects of metal metabolism, and the recent developments in the area of the proteomics of metal transport.     

Centralizing discovery information: from logistics to knowledge at a public organization

Due to the huge amount of data generated in drug discovery programs, their success strongly depends on both the workflows and platforms to manage and, more importantly, to integrate different chemical and biological data sources. At Experimental Therapeutics Program in the Spanish National Cancer Research Center (CNIO), we have addressed our efforts in the design and optimal implementation of those key processes that enable dynamic workflows and interfaces between the different information blocks. Our approach focuses on the development of a common chemical and biological repository (CCBR) that gathers all data that pass quality control criteria. An integral web application (WACBIP) was designed to query against CCBR while providing decision making tools. Currently, our CCBR contains more than 43,000 unique structures as well as experimental data from more than 350 different biological assays. As input sources of the CCBR, we federated a series of Laboratory Information Management Systems (LIMS) which cover sections as follows: chemical synthesis, analytical department, compound logistics, biochemical and cellular data (including high-throughput and high-content screenings; HTS and HCS), computational chemistry (in-silico chemogenomics and physico-chemical profiling) and in-vivo pharmacology. With regard to the last section, an integral In-Vivo Management e-Biobook (IVMB) that handles the entire workflow of in-vivo labs was designed and implemented. Herein we describe the processes and tools that we have developed and implemented, balancing purchase and development, for centralizing discovery information as well as providing decision-making and project management tools - a clear unmet need in public organizations and networks.     

Proteomics and immunomapping of reactive lymph-node and lymphoma

In the present study we show that two-dimensional (2-D) maps together with immuno-detection allow the precise identification of important leukocyte differentiation and tumor markers (e.g., CD3 and CD5), and important cell cycle regulatory molecules such as cyclin dependent kinases, notably CDK6. In addition, the comparative evaluation of molecular expression (e.g., CD5) in maps developed with normal and lymphoma samples can provide reproducible and precise information regarding the molecular expression in different cell populations. Accordingly, we could detect a much increased level of expression of CD5 in mantle cell lymphoma, up to ten times higher than in the control. In addition, CD5 in tumor tissues seems to be microheterogeneous as compared to normal samples.     

Triplet diphenylcarbenes protected by trifluoromethyl and bromine groups. A triplet carbene surviving a day in solution at room temperature

Two types of diphenyldiazomethanes having two trifluoromethyl and two bromine groups at the ortho positions, either in unsymmetrical or in symmetrical fashion, that is, (2,6-dibromo-4-phenylphenyl)[4-phenyl-2,6-bis(trifluoromethyl)phenyl]diazomethane (U-1-N2) and bis(2-bromo-4-phenyl-6-trifluoromethylphenyl)diazomethane (S-1-N2), are prepared. Triplet diphenylcarbenes (U-(3)1 or S-(3)1) are generated from those precursors and are characterized by ESR, UV/vis spectroscopy at low temperature, as well as time-resolved UV/vis spectroscopy at room temperature. Those carbenes are shown to be at least 2 orders of magnitude less reactive than the most stable triplet diphenylcarbene thus far known, that is, bis(2,6-dibromo-4-phenylphenyl)carbene. It has been also shown that S-(3)1 is significantly more stable than U-(3)1 even though both have the same two kinds of substituents. It is suspected that the perpendicular alignment of the two most bulky groups is a more effective way to shield the carbenic center than the planar one. By this way, triplet substituted diphenylcarbene surviving nearly a day in solution at room temperature is realized for the first time.     

Proteomics as a tool for optimization of human plasma protein separation

The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.     

Proteomics as a tool for examining the toxicity of heavy metals

Examining the toxic effects of heavy metals on protein expression can be useful for gaining insight into the biomolecular mechanisms of toxicity and for identifying potential candidate metal-specific protein markers of exposure and response. In this article, we present the state of the art of proteomics in metal-toxicity-related studies. We consider different methods used for sample preparation that depend on the nature of the sample (plants, microorganisms and animals). We also describe different proteomic strategies, both gel-based and gel-free technologies, including two-dimensional gel electrophoresis (2-DE) and multi-dimensional protein-identification technology (MudPIT). We critically review the advantages and the disadvantages of such techniques and discuss the main studies carried out so far. We also comment on future applications and potential research interests within this field.     

Evaluation of TILI-2 as an Anti-Tyrosinase,Anti-Oxidative Agent and Its Role in Preventing Melanogenesis Using a Proteomics Approach

There is a desire to develop new molecules that can combat hyperpigmentation. To this end, the N-terminal cysteine-containing heptapeptide TILI-2 has shown promising preliminary results. In this work, the mechanism by which it works was evaluated using a series of biochemical assays focusing on known biochemical pathways, followed by LC-MS/MS proteomics to discover pathways that have not been considered before. We demonstrate that TILI-2 is a competitive inhibitor of tyrosinase’s monophenolase activity and it could potentially scavenge ABTS and DPPH radicals. It has a very low cytotoxicity up to 1400 µM against human fibroblast NFDH cells and macrophage-like RAW 264.7 cells. Our proteomics study revealed that another putative mechanism by which TILI-2 may reduce melanin production involves the disruption of the TGF-β signaling pathway in mouse B16F1 cells. This result suggests that TILI-2 has potential scope to be used as a depigmenting agent.     

Proteomics and peptidomics in neuroscience. Experience of capabilities and limitations in a neurochemical laboratory

The increasing use of proteomics has created a basis for new strategies to develop methodologies for rapid identification of protein patterns in living organisms. It has also become evident that proteomics has other potential applications than protein and peptide identification, e.g. protein characterization, with the aim of revealing their structure, function(s) and interactions of proteins. In comparative proteomics studies, the protein expression of a certain biological system is compared with another system or the same system under perturbed conditions. Global identification of proteins in neuroscience is extremely complex, owing to the limited availability of biological material and very low concentrations of the molecules. Moreover, in addition to proteins, there are number of peptides that must also be considered in global studies on the central nervous system. In this overview, we focus on and discuss problems related to the different sources of biological material and sample handling, which are part of all preparatory and analytical steps. Straightforward protocols are desirable to avoid excessive purification steps, since loss of material at each step is inevitable. We would like to merge the two worlds of proteomics/peptidomics and neuroscience, and finally we consider different practical and technical aspects, illustrated with examples from our laboratory.     

Natural Product Proteomining,a Quantitative Proteomics Platform,Allows Rapid Discovery of Biosynthetic Gene Clusters for Different Classes of Natural Products

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Longitudinal Bottom-Up Proteomics of Serum,Serum Extracellular Vesicles,and Cerebrospinal Fluid Reveals Candidate Biomarkers for Early Detection of Glioblastoma in a Murine Model

Glioblastoma Multiforme (GBM) is a brain tumor with a poor prognosis and low survival rates. GBM is diagnosed at an advanced stage, so little information is available on the early stage of the disease and few improvements have been made for earlier diagnosis. Longitudinal murine models are a promising platform for biomarker discovery as they allow access to the early stages of the disease. Nevertheless, their use in proteomics has been limited owing to the low sample amount that can be collected at each longitudinal time point. Here we used optimized microproteomics workflows to investigate longitudinal changes in the protein profile of serum, serum small extracellular vesicles (sEVs), and cerebrospinal fluid (CSF) in a GBM murine model. Baseline, pre-symptomatic, and symptomatic tumor stages were determined using non-invasive motor tests. Forty-four proteins displayed significant differences in signal intensities during GBM progression. Dysregulated proteins are involved in cell motility, cell growth, and angiogenesis. Most of the dysregulated proteins already exhibited a difference from baseline at the pre-symptomatic stage of the disease, suggesting that early effects of GBM might be detectable before symptom onset.