Assay bioinformatics

The specificity determinants surrounding the tyrosine phospho-acceptor sites have been determined by various procedures. In PTK assays using various substrates, it was determined that glutamic residues of the N-terminal or C-terminal side of the acceptor are often preferred. The substrate specificity of PTK catalytic domains has been analyzed by peptide library screening for prediction of the optimal peptide substrates. Finally, bioinformatics has been applied to identify phospho-acceptor sites in proteins of PTKs by application of a neural network algorithm. 

At present, there are advanced difference gel electrophoresis (DOGE) Systems and 2-D fluorescence difference gel electrophoresis (2-D DIGE) which enable the analyst to use simultaneously modern (more precise) methods of fluorescent analysis with 2-D electrophoresis (using internal patterns), aided by a fully integrated bioinformatics system. Such systems allow more complete differential protein analysis, while the application of internal standards eliminates differentiation between the intervals, thus ensuring that even the smallest differences will be detected irrespective of the multitude of components. This guarantees reproducibility of results and their statistical reliability. Such assays are one of the platforms employed in the research based on the proteomics method. 

When it comes to the practical details of how bioinformatics can speed the process of drug discovery, it is reasonable to ask what sorts of data could be valuable in that process. The stages of the drug discovery process where bioinformatics makes an impact are target identification, assay selectivity panel selection, and integration throughout the as-... 

Gradually, over the past twenty years, mass spectrometers were interfaced with a number of protein chemistry assays to generate detectors providing superior information. With the increased performance and versatility of the instrumentation dedicated to the life sciences, new analytical strategies for peptide and protein identification and characterization have emerged in which MS and bioinformatic tools are key players. MS has an enormous impact on the capability for structural analysis of bio-molecules, thanks to the ability to create gas phase ions of the peptides and proteins to be analyzed. Peptides and proteins are often charged and polar, making... 

Prior to MS-based substrate specificity assays, certain NRPS substrate specificities can be predicted by bioinformatics. Adenylation domain substrates can be predicted based on their 10 letter code 99,100 by substrate prediction tools such as the NRPS predictor.101 Methyltransferases can be predicted in their substrates and methylation sites by bioinformatic analysis too.102 In addition, substrates of catalytic NRPS domains and tailoring enzymes can be predicted by the structure of the known NRP natural product. Either way, predicted substrates of NRPS domains need to be experimentally verified. A traditional technique to determine substrate specificity of an A domain is the adeonsine triphosphate-pyrophosphate (ATP-PP ) exchange assay. The ATP-PP exchange assay characterizes substrates indirectly by observing the radioactive pyrophosphate incorporation into ATP from a reverse reaction with pyrophosphate and the acyl-adenylate of the substrate.103 Because the PP exchange measures the back exchange of pyrophosphate into ATP, the determined substrate can deviate from the true substrate as it may be only the kinetically most competent substrate of the reverse adenylation reaction. In contrast to this assay, MS has become a more reliable tool to identify NRPS substrates because it determines the true substrate specificity by detection of the complete adenylation reaction product, that is, the substrate tethered on a T domain. 

Quackenbush, J. 2004. Data standards for omic science. Nat. Biotechnol. 22, 613-614. Quackenbush, J. 2005. Using DNA microarrays to assay gene expression. In "Bioinformatics ... 

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