Proteomics 

About: Proteomics is an academic journal published by Wiley. The journal publishes majorly in the area(s): Proteome & Proteomics. It has an ISSN identifier of 1615-9853. Over the lifetime, 7116 publications have been published receiving 347395 citations.

Current two‐dimensional electrophoresis technology for proteomics

Abstract: Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separation of complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where Mr and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Gorg et al., Electrophoresis 2000, 21, 1037-1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5-12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2-DE with IPGs; protein detection and quantitation; computer assisted analysis of 2-DE patterns; protein identification and characterization by MS; two-dimensional protein databases.

Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence.

Abstract: Post-translational modifications (PTMs) occur on almost all proteins analyzed to date. The function of a modified protein is often strongly affected by these modifications and therefore increased knowledge about the potential PTMs of a target protein may increase our understanding of the molecular processes in which it takes part. High-throughput methods for the identification of PTMs are being developed, in particular within the fields of proteomics and mass spectrometry. However, these methods are still in their early stages, and it is indeed advantageous to cut down on the number of experimental steps by integrating computational approaches into the validation procedures. Many advanced methods for the prediction of PTMs exist and many are made publicly available. We describe our experiences with the development of prediction methods for phosphorylation and glycosylation sites and the development of PTM-specific databases. In addition, we discuss novel ideas for PTM visualization (exemplified by kinase landscapes) and improvements for prediction specificity (by using ESS--evolutionary stable sites). As an example, we present a new method for kinase-specific prediction of phosphorylation sites, NetPhosK, which extends our earlier and more general tool, NetPhos. The new server, NetPhosK, is made publicly available at the URL http://www.cbs.dtu.dk/services/NetPhosK/. The issues of underestimation, over-prediction and strategies for improving prediction specificity are also discussed.

Matrigel: A complex protein mixture required for optimal growth of cell culture

Abstract: Numerous cell types require a surface for attachment to grow and proliferate. Certain cells, particularly primary and stem cells, necessitate the use of specialized growth matrices along with specific culture media conditions to maintain the cells in an undifferentiated state. A gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel is commonly used as a basement membrane matrix for stem cells because it retains the stem cells in an undifferentiated state. However, Matrigel is not a well-defined matrix, and therefore can produce a source of variability in experimental results. In this study, we present an in-depth proteomic analysis of Matrigel using a dynamic iterative exclusion method coupled with fractionation protocols that involve ammonium sulfate precipitation, size exclusion chromatography, and one-dimensional SDS-PAGE. The ability to identify the low mass and abundance components of Matrigel illustrates the utility of this method for the analysis of the extracellular matrix, as well as the complexity of the matrix itself.

Comet: An open-source MS/MS sequence database search tool

Abstract: Proteomics research routinely involves identifying peptides and proteins via MS/MS sequence database search. Thus the database search engine is an integral tool in many proteomics research groups. Here, we introduce the Comet search engine to the existing landscape of commercial and open-source database search tools. Comet is open source, freely available, and based on one of the original sequence database search tools that has been widely used for many years.

FunRich: An open access standalone functional enrichment and interaction network analysis tool

Abstract: As high-throughput techniques including proteomics become more accessible to individual laboratories, there is an urgent need for a user-friendly bioinformatics analysis system. Here, we describe FunRich, an open access, standalone functional enrichment and network analysis tool. FunRich is designed to be used by biologists with minimal or no support from computational and database experts. Using FunRich, users can perform functional enrichment analysis on background databases that are integrated from heterogeneous genomic and proteomic resources (>1.5 million annotations). Besides default human specific FunRich database, users can download data from the UniProt database, which currently supports 20 different taxonomies against which enrichment analysis can be performed. Moreover, the users can build their own custom databases and perform the enrichment analysis irrespective of organism. In addition to proteomics datasets, the custom database allows for the tool to be used for genomics, lipidomics and metabolomics datasets. Thus, FunRich allows for complete database customization and thereby permits for the tool to be exploited as a skeleton for enrichment analysis irrespective of the data type or organism used. FunRich (http://www.funrich.org) is user-friendly and provides graphical representation (Venn, pie charts, bar graphs, column, heatmap and doughnuts) of the data with customizable font, scale and color (publication quality).