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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.Isolation of Extracellular Vesicles for Proteomic Profiling
Extracellular vesicles are nano-sized lipid bilayer vesicles released from most cells, including archaea, bacteria, and eukaryotic cells. These membrane vesicles play multiple roles in cell-to-cell communication, including immune modulation, angiogenesis, and transformation of cells by transferring genetic material and functional proteins. They contain specific subsets of proteins, DNA, RNA, and lipids that represent their cellular status. Furthermore, extracellular vesicles are enriched in cell type- or disease-specific vesicular proteins, especially plasma membrane proteins, which have pathophysiological functions; these...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Principles of Protein Labeling Techniques
Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Enrichment of Low-Abundant Protein Targets by Immunoprecipitation Upstream of Mass Spectrometry
Immunoprecipitation (IP) is commonly used upstream of mass spectrometry (MS) as an enrichment tool for low-abundant protein targets. However, several aspects of the classical IP procedure such as nonspecific protein binding to the isolation matrix, detergents or high salt concentrations in wash and elution buffers, and antibody chain contamination in elution fractions render it incompatible with downstream mass spectrometry analysis. Here, we discuss two IP workflows that are designed to minimize or eliminate these contaminants: the first employs biotinylated antibodies and streptavidin magnetic beads while the second meth...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Full-Length Protein Extraction Protocols and Gel-Based Downstream Applications in Formalin-Fixed Tissue Proteomics
Archival formalin-fixed, paraffin-embedded (FFPE) tissue repositories and their associated clinical information can represent a valuable resource for tissue proteomics. In order to make these tissues available for protein biomarker discovery and validation studies, dedicated sample preparation procedures overcoming the intermolecular cross-links introduced by formalin need to be implemented. This chapter describes a full-length protein extraction protocol optimized for downstream gel-based proteomics applications. Using the procedures detailed here, SDS-PAGE, western immunoblotting, GeLC-MS/MS, 2D-PAGE, and 2D-DIGE can be ...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Qualitative and Quantitative Proteomic Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) Tissue
Formalin-fixed, paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the formalin fixation proteins undergo degradation and cross-linking, making conventional protein analysis technologies challenging. Cross-linking is even more challenging when quantitative proteome analysis of FFPE tissue is planned. The use of conventional protein labeling technologies on FFPE tissue has turned out to be problematic as the lysine residue labeling targets are frequently blocked by the formalin treatment. We have established a q...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
De Novo Peptide Structure Prediction: An Overview
Peptide structure identification is an important contribution to the further characterization of the residues involved in functional interactions. De novo structure peptide prediction has, in the past few years, made significant progresses that make reasonable, for peptides up to 50 amino acids, its use for the fast identification of their structural topologies. Here, we introduce some of the concepts underlying approaches of the field, together with their limits. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
Improved Methods for Classification, Prediction, and Design of Antimicrobial Peptides
Peptides with diverse amino acid sequences, structures, and functions are essential players in biological systems. The construction of well-annotated databases not only facilitates effective information management, search, and mining but also lays the foundation for developing and testing new peptide algorithms and machines. The antimicrobial peptide database (APD) is an original construction in terms of both database design and peptide entries. The host defense antimicrobial peptides (AMPs) registered in the APD cover the five kingdoms (bacteria, protists, fungi, plants, and animals) or three domains of life (bacteria, ar...
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
Molecular Modeling of Peptides
This article presents a review of the field of molecular modeling of peptides. The main focus is on atomistic modeling with molecular mechanics potentials. The description of peptide conformations and solvation through potentials is discussed. Several important computer simulation methods are briefly introduced, including molecular dynamics, accelerated sampling approaches such as replica-exchange and metadynamics, free energy simulations and kinetic network models like Milestoning. Examples of recent applications for predictions of structure, kinetics, and interactions of peptides with complex environments are described. ...
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
Building MHC Class II Epitope Predictor Using Machine Learning Approaches
Identification of T-cell epitopes binding to MHC class II molecules is an important step in epitope-based vaccine development. This process has since been accelerated with the use of bioinformatics tools to aid in the prediction of peptide binding to MHC class II molecules and also to systematically scan for candidate peptides in antigenic proteins. There have been many prediction software developed over the years using various methods and algorithms and they are becoming increasingly sophisticated. Here, we illustrate the use of machine learning algorithms to train on MHC class II peptide data represented by feature vecto...
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
Computational Prediction of Short Linear Motifs from Protein Sequences
Short Linear Motifs (SLiMs) are functional protein microdomains that typically mediate interactions between a short linear region in one protein and a globular domain in another. SLiMs usually occur in structurally disordered regions and mediate low affinity interactions. Most SLiMs are 3–15 amino acids in length and have 2–5 defined positions, making them highly likely to occur by chance and extremely difficult to identify. Nevertheless, our knowledge of SLiMs and capacity to predict them from protein sequence data using computational methods has advanced dramatically over the past decade. By considering the b...
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
Brownian Dynamics Simulation of Peptides with the University of Houston Brownian Dynamics (UHBD) Program
This chapter provides the background theory and a practical protocol for performing Brownian dynamics simulation of peptides. Brownian dynamics simulation represents a complementary approach to Monte Carlo and molecular dynamics methods. Unlike Monte Carlo methods, it could provide dynamical information in a timescale longer than the momentum relaxation time. On the other hand, it is faster than molecular dynamics by approximating the solvent by a continuum and by operating in the over-damped limit. This chapter introduces the use of the University of Houston Brownian Dynamics (UHBD) program [1, 2] to perform Brownian dyna...
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
Peptide Toxicity Prediction
Last decade has witnessed the revival of interest in peptides as potential therapeutics candidates. However, one of the bottlenecks in the success of therapeutic peptides in clinics is their toxicity towards eukaryotic cells. Therefore, considerable efforts have been made over the years both in wet and dry lab to overcome this limitation. With the advances in peptide synthesis, now it is possible to fine-tune the physicochemical properties of peptides by incorporating several chemical modifications and thus to optimize the peptide functionality in order to minimize the toxicity without compromising their therapeutic activi...
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
In Silico Design of Antimicrobial Peptides
The rapid spread of drug-resistant pathogenic microbial strains has created an urgent need for the development of new anti-infective molecules, having different mechanism of action in comparison to existing drugs. Natural antimicrobial peptides (AMPs) represent a novel class of molecules with a broad spectrum of activity and a low rate in inducing bacterial resistance. In particular, linear alpha-helical cationic antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement of the innate defense against microbes. However, until now, ma...
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
Synthetic and Structural Routes for the Rational Conversion of Peptides into Small Molecules
The demand for modified peptides with improved stability profiles and pharmacokinetic properties is driving extensive research effort in this field. The conversion of peptides into organic molecules, as traditional drugs, is a long and puzzled way. Many and versatile approaches have been described for designing peptide mimetics: the substitution of natural residues with modified amino acids and the rigidification and modification of the backbone are the main structural and chemical routes walked in medicinal chemistry. All of these strategies have been successfully applied to obtain active new compounds in molecular biolog...
Source: Springer protocols feed by Protein Science - January 1, 2015 Category: Biochemistry Source Type: news
Calculation of Binding Free Energies
Molecular dynamics simulations enable access to free energy differences governing the driving force underlying all biological processes. In the current chapter we describe alchemical methods allowing the calculation of relative free energy differences. We concentrate on the binding free energies that can be obtained using non-equilibrium approaches based on the Crooks Fluctuation Theorem. Together with the theoretical background, the chapter covers practical aspects of hybrid topology generation, simulation setup, and free energy estimation. An important aspect of the validation of a simulation setup is illustrated by mean...
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
