The Whereabouts of 2D Gels in Quantitative Proteomics

Two-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using 2D gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of 2D gels is performed

Membrane proteins and proteomics: Love is possible, but so difficult

Despite decades of extensive research, the large-scale analysis of membrane proteins remains a difficult task. This is due to the fact that membrane proteins require a carefully balanced hydrophilic and lipophilic environment, which optimum varies with different proteins, while most protein chemistry methods work mainly, if not only, in water-based media. Taking this review [Santoni, Molloy and Rabilloud, Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000, 21, 1054-1070] as a pivotal paper, the current paper analyzes how the field of membrane proteomics exacerbated the trend in proteomics, i.e. developing alternate methods to the historical two-dimensional electrophoresis, and thus putting more and more pressure on the mass spectrometry side. However, in the case of membrane proteins, the incentive in doing so is due to the poor solubility of membrane proteins. This review also shows that in some situations, where this solubility problem is less acute, two-dimensional electrophoresis remains a method of choice. Last but not least, this review also critically examines the alternate approaches that have been used for the proteomic analysis of membrane proteins

Power and limitations of electrophoretic separations in proteomics strategies

Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed

Two-dimensional gel electrophoresis in proteomics: past, present and future

Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned

Proteome signatures-how are they obtained and what do they teach us?

The dawn of a new Proteomics era, just over a decade ago, allowed for large-scale protein profiling studies that have been applied in the identification of distinctive molecular cell signatures. Proteomics provides a powerful approach for identifying and studying these multiple molecular markers in a vast array of biological systems, whether focusing on basic biological research, diagnosis, therapeutics, or systems biology. This is a continuously expanding field that relies on the combination of different methodologies and current advances, both technological and analytical, which have led to an explosion of protein signatures and biomarker candidates. But how are these biological markers obtained? And, most importantly, what can we learn from them? Herein, we briefly overview the currently available approaches for obtaining relevant information at the proteome level, while noting the current and future roles of both traditional and modern proteomics. Moreover, we provide some considerations on how the development of powerful and robust bioinformatics tools will greatly benefit high-throughput proteomics. Such strategies are of the utmost importance in the rapidly emerging field of immunoproteomics, which may play a key role in the identification of antigens with diagnostic and/or therapeutic potential and in the development of new vaccines. Finally, we consider the present limitations in the discovery of new signatures and biomarkers and speculate on how such hurdles may be overcome, while also offering a prospect for the next few years in what could be one of the most significant strategies in translational medicine research.FCT and COMPETE grants PTDC/BIA-MIC/113450/2009, FCOMP-01-0124-FEDER- 014309, QOPNA research unit (project PEst-C/QUI/UI0062/2013), iBiMED (UID/BIM/04501/2013) RNEM (National Mass Spectrome- try Network), and CENTRO-07-ST24-FEDER-002034. The authors also thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project B BioHealth - Biotechnology and Bioengineering ap- proaches to improve health quality ^ , Ref. NORTE-07-0124-FEDER- 000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. Project "Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB", Ref.FCOMP-01-0124-FEDER-02746

Use of systems biology to decipher host–pathogen interaction networks and predict biomarkers

AbstractIn systems biology, researchers aim to understand complex biological systems as a whole, which is often achieved by mathematical modelling and the analyses of high-throughput data. In this review, we give an overview of medical applications of systems biology approaches with special focus on host–pathogen interactions. After introducing general ideas of systems biology, we focus on (1) the detection of putative biomarkers for improved diagnosis and support of therapeutic decisions, (2) network modelling for the identification of regulatory interactions between cellular molecules to reveal putative drug targets and (3) module discovery for the detection of phenotype-specific modules in molecular interaction networks. Biomarker detection applies supervised machine learning methods utilizing high-throughput data (e.g. single nucleotide polymorphism (SNP) detection, RNA-seq, proteomics) and clinical data. We demonstrate structural analysis of molecular networks, especially by identification of disease modules as a novel strategy, and discuss possible applications to host–pathogen interactions. Pioneering work was done to predict molecular host–pathogen interactions networks based on dual RNA-seq data. However, currently this network modelling is restricted to a small number of genes. With increasing number and quality of databases and data repositories, the prediction of large-scale networks will also be feasible that can used for multidimensional diagnosis and decision support for prevention and therapy of diseases. Finally, we outline further perspective issues such as support of personalized medicine with high-throughput data and generation of multiscale host–pathogen interaction models

Characterizing protein compartmentalization of plant energy metabolism

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Transforming growth factor-beta targets Formin-like 2 for Angiopoietin-like 4 secretion during the epithelial mesenchymal transition

Epithelial to Mesenchymal transition (EMT) is a highly dynamic process that plays a crucial role in tumor progression and metastasis. While remodelling of the actin cytoskeleton is a hallmark of EMT, the responsible actin regulating factors are less well understood. Formins are involved in numerous cellular mechanisms, ranging from cytokinesis to cell adhesion and motility. The Rho-GTPase effectors of the formin family compromise the largest group of actin nucleators and are emerging as relevant pharmacological targets. A critical role of Formin-like 2 (FMNL2) in the assembly of junctional actin at newly forming cell-cell contacts in a 3D matrix has been described. This activity originates downstream of Rac1 and is in line with a physical association of FMNL2 and components of the cadherin-catenin complex. FMNL2 was further recently implicated in β1-integrin trafficking as a direct PKC target required for cancer cell invasion. Here we found that transforming growth factor-beta (TGFβ)-driven EMT leads to an upregulation of PKC resulting in the phosphorylation and activation of FMNL2 in epithelial cells. Proteomic screening for TGFβ-mediated phospho-FMNL2 binding partners identified the tumor promotor ANGPTL4 as a specific binding partner. ANGPTL4 has important roles in cancer development and progression including promoting invasion and metastasis. We found that FMNL2 and ANGPTL4 directly interact under TGF-induced EMT. Our data show that FMNL2 is a critical regulator of ANGPTL4 secretion. Secretion of ANGPTL4 is diminished upon loss of FMNL2 and its phosphorylation. We further observed that ANGPTL4 is sequestered in the Golgi apparatus colocalizing with markers of the trans-Golgi network. Live imaging of vesicle secretion from the Golgi confirmed the transient co-localization of ANGPTL4 and FMNL2. Moreover, ANGPTL4 and FMNL2 modulate cell-cell contact integrity and ANGPTL4 silenced cells fail to disassemble their underlying cell-cell contacts to execute EMT. This effect was further enhanced upon FMNL2 knockout using FMNL2 CRISPR/Cas9 cell line. However, re-introduction of ANGPTL4 restored the mesenchymal phenotype and prompted the dissolution of cell-cell adhesions. Finally, we found that cellular invasion promoted by TGFβ depends on FMNL2 and is reduced upon ANGPTL4 silencing. Taken together, our data point towards a crucial role of FMNL2 for EMT via ANGPTL4 secretion

Expression dynamics of the hepatic mitochondrial proteome of the Sod2+/- mouse in response to troglitazone administration

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