Springer protocols feed by Protein Science 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Identification of DNA Damage Checkpoint-Dependent Protein Interactions in Saccharomyces cerevisiae Using Quantitative Mass Spectrometry
The DNA damage checkpoint (DDC) is an evolutionarily conserved signaling pathway that is crucial to maintain genomic integrity. In response to DNA damage, DDC kinases are rapidly activated and phosphorylate an elaborate network of substrates involved in multiple cellular processes. An important role of the DDC response is to assemble protein complexes. However, for most of the DDC substrates, how the DDC-dependent phosphorylation modulates their network of interactions remains to be established. Here, we present a protocol for the identification of DDC-dependent protein–protein interactions based on Stable Isotope La...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Bioinformatics for Proteomics: Opportunities at the Interface Between the Scientists, Their Experiments, and the Community
Within the last decade, bioinformatics has moved from command line scripts dedicated to single experiments towards production grade software integrated in experimental workflows providing a rich environment for biological investigation. Located at the interface between the scientists, their experiments, and the community, bioinformatics acts as a gateway to a wide source of information. This chapter does not list tools and methods, but rather hints at how bioinformatics can help in improving biological projects, all the way from their initial design to the dissemination of the results. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Quantification of Proteins by Label-Free LC-MSE
We present a descriptive protocol for label-free quantitation of proteins by one DIA method termed LC-MSE, which facilitates large-scale quantification of proteins without the need for isotopic labelling and with no theoretical limit to the number of samples included in an experiment. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Spectral Counting Label-Free Proteomics
Label-free proteome quantification methods used in bottom-up mass-spectrometry based proteomics are gaining more popularity as they are easy to apply and can be integrated into different workflows without any extra effort or cost. In the label-free proteome quantification approach, samples of interest are prepared and analyzed separately. Mass-spectrometry is generally not recognized as a quantitative method as the ionization efficiency of peptides is dependent on composition of peptides. Label-free quantification methods have to overcome this limitation by additional computational calculations. There are several algorithm...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Employing TMT Quantification in Shotgun-MS Proteomic Analysis: A Focus on Skeletal Muscle
Quantify proteins present in a sample is the ultimate goal of a proteomic experiment. Although it might reveal countless physiological and /or pathological process on a studied model, it is still a daunting task to perform. Of the protein present in a sample, only a small number can be identified and even a lesser number quantified, each with its own weaknesses and strengths. Presented here are the “tandem mass tags” isobaric labels (TMT) and a protocol for its utilization in the quantification of skeletal muscle, which might be adapted also to other animal tissues. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Employing TMT Quantification in a Shotgun-MS Platform
The blood serum proteome may be an ideal source of disease biomarkers, although its complexity necessitates novel strategies to enrich and quantify lower-abundance proteins with biomarker utility. Herein, serum samples from pre-diagnosis pancreatic cancer cases and controls were compared using a workflow of immunodepletion, multi-lectin fractionation, and peptide tandem mass tag (TMT) labeling. Samples were then subjected to SCX and high pH reversed-phase fractionation and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The aim was the discovery of candidate serum biomarkers of pancreatic cancer, alt...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Brain Quantitative Proteomics Combining GeLC-MS and Isotope-Coded Protein Labeling (ICPL)
Proteomics has been revolutionized by the rapid advance of mass spectrometric instrumentations and techniques. Parallel methodologies for the quantification of proteomes also evolved, including in vitro stable isotope labeling. Here, we present a protocol for employing isotope-coded protein labeling (ICPL) as part of a shotgun proteomics workflow denoting its advantages and disadvantages. This protocol is suitable to studying any proteome of interest, only requiring a specific sample preparation and protein identification. Given our expertise, descriptions here are centered on the study of brain disorders. (Source: Springe...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Determining Protein Subcellular Localization in Mammalian Cell Culture with Biochemical Fractionation and iTRAQ 8-Plex Quantification
Protein subcellular localization is a fundamental feature of posttranslational functional regulation. Traditional microscopy based approaches to study 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 workflow coupling biochemical fractionation and iTRAQ™ 8-plex quantification, can be applied for the high-throughput characterization of protein localization in a mammalian ce...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Analysis of Individual Protein Turnover in Live Animals on a Proteome-Wide Scale
Classical quantitative proteomics studies focus on the relative or absolute concentration of proteins at a given time. In contrast, the investigation of 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 proteins allows detecting functional relationships and common regulation mechanisms. Moreover, comparing turnover of individual brain and plasma proteins between control- and treatment-gr...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Stable Isotope Labeling in Mammals (SILAM)
Analysis of animal models of disease is essential to the understanding of human disease and the identification of potential targets for clinical drugs. Global analysis of proteins by mass spectrometry is an important tool for these studies. Stable isotope labeling in mammals (SILAM) was developed to quantitate the proteomes of rodents using mass spectrometry. The crux of SILAM analysis is the complete labeling of all proteins in a rodent with heavy nitrogen (15N). These 15N tissues are then employed as an internal standard for quantitative proteomics analysis using a high-resolution and mass-accuracy mass spectrometer. (So...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Analysis of Proteome Dynamics in Mice by Isotopic Labeling
Recent advances in mass spectrometry and in vivo isotopic labeling have enabled proteome-wide analyses of protein turnover in complex organisms. Here, we describe a protocol for analyzing protein turnover rates in mouse tissues by comprehensive 15N labeling. The procedure involves the complete isotopic labeling of blue green algae (Spirulina platensis) with 15N and utilizing it as a source of dietary nitrogen for mice. We outline a detailed protocol for in-house production of 15N-labeled algae, labeling of mice, and analysis of isotope incorporation kinetics by mass spectrometry. The methodology can be adapted to analyze p...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
SILAC Yeast: From Labeling to Comprehensive Proteome Quantification
Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex mixtures, enabling characterization and comparison 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 single cells to entire multicellular organisms. In this chapter, detailed instructions for SILAC labeling yeast are provided, including a series of quality checks for evaluating la...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
IPG Strip-Based Peptide Fractionation for Shotgun Proteomics
Efficient fractionation of peptides is an essential prerequisite for comprehensive analysis of complex protein mixtures by shotgun mass spectrometry. The 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 is highly reproducible. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
GeLC-MS/MS Analysis of Complex Protein Mixtures
Discovery-based proteomics has found its place in nearly every facet of biological research. A key objective of this approach is to maximize sequence coverage 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 electrophoresis followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) is a robust and reproducible method for qualitative and quantitative proteomic analysis. Here we...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Fully Automatable Multidimensional Reversed-Phase Liquid Chromatography with Online Tandem Mass Spectrometry
Liquid chromatography (LC) is essential for sample fractionation in shotgun proteomics applications. With suitable design, common LC separation chemistries, 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 system that is compatible with deep proteome profiling on common shotgun proteomics platforms. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
