Titlebook: Shotgun Proteomics; Methods and Protocol Daniel Martins-de-Souza Book 2014 Springer Science+Business Media New York 2014 High throughput id

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LC-MALDI-TOF/TOF for Shotgun Proteomicsures in shotgun proteomics experiments. Approaches involving the use of matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI-MS/MS) comprise the preparation of protein extracts, their enzymatic digestion, the separation of the resulting peptides by nanoscale liquid chromatography

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Fully Automatable Multidimensional Reversed-Phase Liquid Chromatography with Online Tandem Mass Spectries, including reversed-phase (RP) and strong cation exchange (SCX) mode, can be combined in online multidimensional LC to greatly enhance the overall separation power and, thus, proteome coverage. This protocol describes the design and assembly of a flexible online multidimensional RP-SCX-RP LC s

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GeLC-MS/MS Analysis of Complex Protein Mixturescoverage for proteins across a wide concentration range. Fractionating samples at the protein level is one of the most common ways to circumvent challenges due to sample complexity and improve proteome coverage. Of the available methods, one-dimensional sodium dodecyl sulfate-polyacrylamide gel elec

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IPG Strip-Based Peptide Fractionation for Shotgun ProteomicsThe separation of peptides by isoelectric focusing is particularly attractive due to its orthogonality to reverse-phase HPLC. Here, we present a protocol for in-gel peptide isoelectric focusing using immobilized pH gradient strips. The method shows high resolving power for up to 1 mg of sample and i

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SILAC Yeast: From Labeling to Comprehensive Proteome Quantificationparison of cellular functional states in a proteome-wide scale. In this context, stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a simple yet powerful approach, which has been applied to address different biological questions across a variety of systems, ranging from

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Analysis of Proteome Dynamics in Mice by Isotopic Labeling we describe a protocol for analyzing protein turnover rates in mouse tissues by comprehensive .N labeling. The procedure involves the complete isotopic labeling of blue green algae (.) with .N and utilizing it as a source of dietary nitrogen for mice. We outline a detailed protocol for in-house pro

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Analysis of Individual Protein Turnover in Live Animals on a Proteome-Wide Scaleof protein turnover reveals the dynamics leading to these states. Analyzing the balance between synthesis and degradation of individual proteins provides insights into the regulation of protein concentration and helps understanding underlying biological processes. Comparing the half-lives of protein

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Determining Protein Subcellular Localization in Mammalian Cell Culture with Biochemical Fractionatio protein localization are typically of limited throughput, and dependent on the availability of antibodies with high specificity and sensitivity, or fluorescent fusion proteins. In this chapter we describe how Localization of Organelle Proteins by Isotope Tagging (LOPIT), a mass spectrometry based w

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