Methods in molecular biology
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Cell signaling and functions heavily rely on post-translational modifications (PTMs) of proteins. Their high-throughput characterization is thus of utmost interest for multiple biological and medical investigations. ⋯ However, the large and complex datasets produced pose multiple data interpretation challenges, ranging from spectral interpretation to statistical and multivariate analyses. Here, we present a typical workflow to interpret such data.
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The activated partial thromboplastin time (APTT) is a useful global assay for the assessment of the contact factor pathway of hemostasis and its inhibitors. The test is usually performed on fully automated analyzers using commercially prepared reagents. The three main clinical areas of interest are detection of factor deficiencies, detection of lupus anticoagulants and in the monitoring of therapy with unfractionated heparin. Methods are described here for assessing APTT reagents for their sensitivity to clotting time prolongation in each of these areas of interest.
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Comparative profiling proteomics experiments are important tools in biological research. In such experiments, tens to hundreds of thousands of peptides are measured simultaneously, with the goal of inferring protein abundance levels. ⋯ Previously we have reported the non-normal distribution of SILAC datasets, and demonstrated the permutation test to be a superior method for the statistical evaluation of non-normal peptide ratios. This chapter outlines the steps and the R scripts that can be used for performing permutation analysis with false discovery rate control via the Benjamini-Yekutieli method.
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Post-translational modifications (PTMs) are one of the main contributors to the diversity of proteoforms in the proteomic landscape. In particular, protein phosphorylation represents an essential regulatory mechanism that plays a role in many biological processes. Protein kinases, the enzymes catalyzing this reaction, are key participants in metabolic and signaling pathways. ⋯ In this chapter, we demonstrate two text mining tools, RLIMS-P and eFIP, for the retrieval and extraction of kinase-substrate-site data and phosphorylation-dependent PPIs from the literature. These tools offer several advantages over a literature search in PubMed as their results are specific for phosphorylation. RLIMS-P and eFIP results can be sorted, organized, and viewed in multiple ways to answer relevant biological questions, and the protein mentions are linked to UniProt identifiers.
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Recent advances in mass spectrometry based proteomic techniques and publicly available large proteomic repositories are being exploited to characterize the proteome of multiple organisms. While humongous amount of proteomic data is being acquired and analyzed, many biological questions still remain unanswered. Proteotypic peptides which uniquely represent target proteins or a protein isoform are used as an alternative strategy for protein identification in the field of immunological methods and targeted proteomic techniques. Using different computational approaches, resources and techniques used in the identification of proteotypic peptides of target proteins is discussed here.