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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.Structural Biology of Nonribosomal Peptide Synthetases
The nonribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain tha...
Source: Springer protocols feed by Protein Science - February 6, 2016 Category: Biochemistry Source Type: news
Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi
Filamentous fungi are historically known as rich sources for production of biologically active natural products, so-called secondary metabolites. One particularly pharmaceutically relevant chemical group of secondary metabolites is the nonribosomal peptides synthesized by nonribosomal peptide synthetases (NRPSs). As most of the fungal NRPS gene clusters leading to production of the desired molecules are not expressed under laboratory conditions, efforts to overcome this impediment are crucial to unlock the full chemical potential of each fungal species. One way to activate these silent clusters is by overexpressing and del...
Source: Springer protocols feed by Protein Science - February 6, 2016 Category: Biochemistry Source Type: news
Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes
During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this “traditional” cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural ami...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Testing Suitability of Cell Cultures for SILAC-Experiments Using SWATH-Mass Spectrometry
Precise quantification is a major issue in contemporary proteomics. Both stable-isotope-labeling and label-free methods have been established for differential protein quantification and both approaches have different advantages and disadvantages. The present protocol uses the superior precision of label-free SWATH-mass spectrometry to test for suitability of cell lines for a SILAC-labeling approach as systematic regulations may be introduced upon incorporation of the “heavy” amino acids. The SILAC-labeled cell cultures can afterwards be used for further analyses where stable-isotope-labeling is mandatory or has...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Targeted Phosphoproteome Analysis Using Selected/Multiple Reaction Monitoring (SRM/MRM)
Mass spectrometry-based phosphoproteomics has been rapidly spread based on the advancement of mass spectrometry and development of efficient enrichment techniques for phosphorylated proteins or peptides. Non-targeted approach has been employed in most of the studies for phosphoproteome analysis. However, targeted approach using selected/multiple reaction monitoring (SRM/MRM) is an indispensible technique used for the quantitation of known targets especially when we have many samples to quantitate phosphorylation events on proteins in biological or clinical samples. We herein describe the application of a large-scale phosph...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses
When deciding to perform a quantitative proteomics analysis, selectivity, sensitivity, and reproducibility are important criteria to consider. The use of multiple reaction monitoring (MRM) has emerged as a powerful proteomics technique in that regard since it avoids many of the problems typically observed in discovery-based analyses. A prerequisite for such a targeted approach is that the protein targets are known, either as a result of previous global proteomics experiments or because a specific hypothesis is to be tested. When guidelines that have been established in the pharmaceutical industry many decades ago are taken...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Monitoring PPARG-Induced Changes in Glycolysis by Selected Reaction Monitoring Mass Spectrometry
As cells develop and differentiate, they change in function and morphology, which often precede earlier changes in signaling and metabolic control. Here we present a selected reaction monitoring (SRM) approach which allows for the parallel quantification of metabolic regulators and their downstream targets. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Selected Reaction Monitoring to Measure Proteins of Interest in Complex Samples: A Practical Guide
Biology and especially systems biology projects increasingly require the capability to detect and quantify specific sets of proteins across multiple samples, for example the components of a biological pathway through a set of perturbation-response experiments. Targeted proteomics based on selected reaction monitoring (SRM) has emerged as an ideal tool to this purpose, and complements the discovery capabilities of shotgun proteomics methods. SRM experiments rely on the development of specific, quantitative mass spectrometric assays for each protein of interest and their application to the quantification of the protein set i...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Two Birds with One Stone: Parallel Quantification of Proteome and Phosphoproteome Using iTRAQ
Altered and abnormal levels of proteins and their phosphorylation states are associated with many disorders. Detection and quantification of such perturbations may provide a better understanding of pathological conditions and help finding candidates for treatment or biomarkers. Over the years, isobaric mass tags for relative quantification of proteins and protein phosphorylation by mass spectrometry have become increasingly popular. One of the most commonly used isobaric chemical tags is iTRAQ (isobaric tag for relative and absolute quantitation). In a typical iTRAQ-8plex experiment, a multiplexed sample amounts for up to ...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Sample Preparation Approaches for iTRAQ Labeling and Quantitative Proteomic Analyses in Systems Biology
Among a variety of global quantification strategies utilized in mass spectrometry (MS)-based proteomics, isobaric tags for relative and absolute quantitation (iTRAQ) are an attractive option for examining the relative amounts of proteins in different samples. The inherent complexity of mammalian proteomes and the diversity of protein physicochemical properties mean that complete proteome coverage is still unlikely from a single analytical method. Numerous options exist for reducing protein sample complexity and resolving digested peptides prior to MS analysis. Indeed, the reliability and efficiency of protein identificatio...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Systemic Analysis of Regulated Functional Networks
In biological and medical sciences, high throughput analytical methods are now commonly used to investigate samples of different conditions, e.g., patients versus controls. Systemic functional analyses emerged as a reference method to go beyond a list of regulated compounds, and identify activated or inactivated biological functions. This approach holds the promise for a better understanding of biological systems, of the mechanisms involved in disease progression, and thus improved diagnosis, prognosis, and treatment. In this chapter, we present a simple workflow to conduct pathway analyses on biological data using the fre...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
A Simple Workflow for Large Scale Shotgun Glycoproteomics
Targeting subproteomes is a good strategy to decrease the complexity of a sample, for example in body fluid biomarker studies. Glycoproteins are proteins with carbohydrates of varying size and structure attached to the polypeptide chain, and it has been shown that glycosylation plays essential roles in several vital cellular processes, making glycosylation a particularly interesting field of study. Here, we describe a method for the enrichment of glycosylated peptides from trypsin digested proteins in human cerebrospinal fluid. We also describe how to perform the data analysis on the mass spectrometry data for such samples...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Multiplexed Quantitative Proteomics for High-Throughput Comprehensive Proteome Comparisons of Human Cell Lines
The proteome is the functional entity of the cell, and perturbations of a cellular system almost always cause changes in the proteome. These changes are a molecular fingerprint, allowing characterization and a greater understanding of the effect of the perturbation on the cell as a whole. Monitoring these changes has therefore given great insight into cellular responses to stress and disease states, and analytical platforms to comprehensively analyze the proteome are thus extremely important tools in biological research. Mass spectrometry has evolved as the most relevant technology to characterize proteomes in a comprehens...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Interpretation of Quantitative Shotgun Proteomic Data
In quantitative proteomics, large lists of identified and quantified proteins are used to answer biological questions in a systemic approach. However, working with such extensive datasets can be challenging, especially when complex experimental designs are involved. Here, we demonstrate how to post-process large quantitative datasets, detect proteins of interest, and annotate the data with biological knowledge. The protocol presented can be achieved without advanced computational knowledge thanks to the user-friendly Perseus interface (available from the MaxQuant website,
www.maxquant.org
...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
From Phosphoproteome to Modeling of Plant Signaling Pathways
Quantitative proteomic experiments in recent years became almost routine in many aspects of biology. Particularly the quantification of peptides and corresponding phosphorylated counterparts from a single experiment is highly important for understanding of dynamics of signaling pathways. We developed an analytical method to quantify phosphopeptides (pP) in relation to the quantity of the corresponding non-phosphorylated parent peptides (P). We used mixed-mode solid-phase extraction to purify total peptides from tryptic digest and separated them from most of the phosphorous-containing compounds (e.g., phospholipids, nucleot...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
