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Since its inception, the yeast two-hybrid (Y2H) system has proven to be an efficient system to identify novel protein-protein interactions. However, Y2H screens are sometimes criticized for generating high rates of false-positives. Minimizing false-positive interactions is especially important in proteome wide high-throughput (HT) Y2H. Here, we summarize various approaches that reduce false-positives...
As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We...
Proteomics and functional genomics are emerging new research fields devoted to the study of the entire collection of proteins and mRNA transcripts (collectively known as gene products) that define a biological system. DNA microarrays are now a popular platform for measuring changes in messenger RNA transcript levels on a genome-wide scale, while gel-free shotgun profiling methods based on tandem mass...
Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS–PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein...
High throughput protein analysis (i.e., proteomics) first became possible when sensitive peptide mass mapping techniques were developed, thereby allowing for the possibility of identifying and cataloging most 2D gel electrophoresis spots. Shortly thereafter a few groups pioneered the idea of identifying proteins by using peptide tandem mass spectra to search protein sequence databases. Hence, it became...
This tutorial article introduces mass spectrometry (MS) for peptide fragmentation and protein identification. The current approaches being used for protein identification include top-down and bottom-up sequencing. Top-down sequencing, a relatively new approach that involves fragmenting intact proteins directly, is briefly introduced. Bottom-up sequencing, a traditional approach that fragments peptides...
Multidimensional liquid chromatography techniques have been coupled to tandem mass spectrometry to provide a robust method to identify proteins in complex mixtures. Data acquisition is interfaced directly with search algorithms for identification through cross-correlation with databases. This review describes the most recent advances in methodologies for protein identification by mass spectrometry...
Discovery of novel protein biomarkers is essential for successful drug discovery and development. These novel protein biomarkers may aid accelerated drug efficacy, response, or toxicity decision making based on their enhanced sensitivity and/or specificity. These biomarkers, if necessary, could eventually be converted into novel diagnostic marker assays. Proteomic platforms developed over the past...
Aptamers are nucleic acid species that are selected in vitro for their specific binding properties. We describe methods for the production and processing of aptamer microarrays, including detailed procedures for the high-throughput, enzymatic synthesis of 5′ RNA biotinylated aptamers and for arraying them onto streptavidin-coated glass slides. Also presented are methods for processing the aptamer...
Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because...
A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies...
The preparation of large quantities of purified membrane proteins for structural studies presents significant difficulties. Central among these are the frequent toxicity associated with over-expressing membrane targets and the difficulty associated with identifying the appropriate detergents for their solubilization and purification. To begin addressing these challenges, and lay the groundwork for...
Chaperonin (Cpn)60 proteins have the ability to activate human and murine myeloid cells. There is contradictory evidence that the receptor for this protein is either similar to that of lipopolysaccharide—CD14 and one or other toll-like receptor (e.g. TLR4) or is some other, undidentified, receptor. In an attempt to directly identify the receptor for Mycobacterium tuberculosis Cpn60.1 we have used...
While the analysis of the cartilage proteome is important for our comprehensive understanding of the development and disease of this important tissue, several unique features of cartilage present some technical obstacles. Firstly, cartilage is difficult to obtain in adequate quantities for many protein analyses, especially from mice which are otherwise powerful experimental models. Furthermore, the...
Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory...
Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology...
To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass...
Gene trapping is a high-throughput insertional mutagenesis approach that has been primarily used in mouse embryonic stem cells (ESCs). As a high throughput technology, gene trapping helped to generate tenth of thousands of ESC lines harboring mutations in single genes that can be used for making knock-out mice. Ongoing international efforts operating under the umbrella of the International Knockout...
Large-scale proteomic screens are increasingly employed for placing genes into specific pathways. Therefore generic methods providing a physiological context for protein–protein interaction studies are of great interest. In recent years many protein–protein interactions have been determined by affinity purification followed by mass spectrometry (AP–MS). Among many different AP–MS approaches, the recently...
Spectral library searching is an emerging approach in peptide identifications from tandem mass spectra, a critical step in proteomic data analysis. In spectral library searching, a spectral library is first meticulously compiled from a large collection of previously observed peptide MS/MS spectra that are conclusively assigned to their corresponding amino acid sequence. An unknown spectrum is then...
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