Methods in molecular biology
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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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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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In this chapter we describe the workflow we use for labeled quantitative proteomics analysis using tandem mass tags (TMT) starting with the sample preparation and ending with the multivariate analysis of the resulting data. We detail the step-by-step process from sample processing, labeling, fractionation, and data processing using Proteome Discoverer through to data analysis and interpretation in the context of a multi-run experiment. The final analysis and data interpretation rely on an R package we call TMTPrepPro, which are deployed on a local GenePattern server, and used for generating various outputs which are also outlined herein.
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A true and accurate bottom-up global proteomic measurement will only be achieved when all proteins in a sample can be digested efficiently and at least some peptides recovered on which to base an estimate of abundance. Integral membrane proteins make up around one-third of the proteome and require specialized protocols if they are to be successfully solubilized for efficient digestion by the enzymes used in bottom-up proteomics. ⋯ A subset of peptides is purified by reverse-phase solid-phase extraction and fractionated by strong-cation exchange prior to nano-liquid chromatography with data-dependent tandem mass spectrometry. For quantitative proteomics experiments a protocol is described for stable-isotope coding of peptides using dimethylation of primary amines allowing for three-way sample multiplexing.
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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.