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.A Proteomic Workflow Using High-Throughput De Novo Sequencing Towards Complementation of Genome Information for Improved Comparative Crop Science
The proteomic study of non-model organisms, such as many crop plants, is challenging due to the lack of comprehensive genome information. Changing environmental conditions require the study and selection of adapted cultivars. Mutations, inherent to cultivars, hamper protein identification and thus considerably complicate the qualitative and quantitative comparison in large-scale systems biology approaches. With this workflow, cultivar-specific mutations are detected from high-throughput comparative MS analyses, by extracting sequence polymorphisms with de novo sequencing. Stringent criteria are suggested to filter for conf...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Generating Sample-Specific Databases for Mass Spectrometry-Based Proteomic Analysis by Using RNA Sequencing
Mass spectrometry-based methods allow for the direct, comprehensive analysis of expressed proteins and their quantification among different conditions. However, in general identification of proteins by assigning experimental mass spectra to peptide sequences of proteins relies on matching mass spectra to theoretical spectra derived from genomic databases of organisms. This conventional approach limits the applicability of proteomic methodologies to species for which a genome reference sequence is available. Recently, RNA-sequencing (RNA-Seq) became a valuable tool to overcome this limitation by de novo construction of data...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Profiling of Small Molecules by Chemical Proteomics
Chemical proteomics provides a powerful means to gain systems-level insight into the mode of action of small molecules and/or natural products. In contrast to high-throughput screening efforts which only interrogate selected subproteomes such as kinases and often only consider individual domains, the methodology presented herein allows for the determination of the molecular targets of small molecules or drugs in a more relevant physiological setting. As such, the compound of interest is exposed to the entire variety of cellular proteins considering all naturally occurring posttranslational modifications and activation stat...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
A Systems Approach to Understand Antigen Presentation and the Immune Response
The mammalian immune system has evolved to respond to pathogenic, environmental, and cellular changes in order to maintain the health of the host. These responses include the comparatively primitive innate immune response, which represents a rapid and relatively nonspecific reaction to challenge by pathogens and the more complex cellular adaptive immune response. This adaptive response evolves with the pathogenic challenge, involves the cross talk of several cell types, and is highly specific to the pathogen due to the liberation of peptide antigens and their presentation on the surface of affected cells. Together these tw...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Simple and Effective Affinity Purification Procedures for Mass Spectrometry-Based Identification of Protein-Protein Interactions in Cell Signaling Pathways
Identification of protein-protein interactions can be a critical step in understanding the function and regulation of a particular protein and for exploring intracellular signaling cascades. Affinity purification coupled to mass spectrometry (APMS) is a powerful method for isolating and characterizing protein complexes. This approach involves the tagging and subsequent enrichment of a protein of interest along with any stably associated proteins that bind to it, followed by the identification of the interacting proteins using mass spectrometry. The protocol described here offers a quick and simple method for routine sample...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Characterization of Protein N-Glycosylation by Analysis of ZIC-HILIC-Enriched Intact Proteolytic Glycopeptides
Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost ...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Ethyl Esterification for MALDI-MS Analysis of Protein Glycosylation
Ethyl esterification is a technique for the chemical modification of sialylated glycans, leading to enhanced stability when performing matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), as well as allowing the efficient detection of both sialylated and non-sialylated glycans in positive ion mode. In addition, the method shows specific reaction products for α2,3- and α2,6-linked sialic acids, leading to an MS distinguishable mass difference. Here, we describe the ethyl esterification protocol for 96 glycan samples, including enzymatic N-glycan release, the aforementioned ethyl esterifica...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Tissue MALDI Mass Spectrometry Imaging (MALDI MSI) of Peptides
Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a technique to visualize molecular features of tissues based on mass detection. This chapter focuses on MALDI MSI of peptides and provides detailed operational instructions for sample preparation of cryoconserved and formalin-fixed paraffin-embedded (FFPE) tissue. Besides sample preparation we provide protocols for the MALDI measurement, tissue staining, and data analysis. On-tissue digestion and matrix application are described for two different commercially available and commonly used spraying devices: the SunCollect (SunChrom) and the I...
Source: Springer protocols feed by Protein Science - December 29, 2015 Category: Biochemistry Source Type: news
Differential Analysis of 2-D Maps by Pixel-Based Approaches
Two approaches to the analysis of 2-D maps are available: the first one involves a step of spot detection on each gel image; the second one is based instead on the direct differential analysis of 2-D map images, following a pixel-based procedure. Both approaches strongly depend on the proper alignment of the gel images, but the pixel-based approach allows to solve important drawbacks of the spot-volume procedure, i.e., the problem of missing data and of overlapping spots. However, this approach is quite computationally intensive and requires the use of algorithms able to separate the information (i.e., spot-related informa...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Nonlinear Dimensionality Reduction by Minimum Curvilinearity for Unsupervised Discovery of Patterns in Multidimensional Proteomic Data
Dimensionality reduction is largely and successfully employed for the visualization and discrimination of patterns, hidden in multidimensional proteomics datasets. Principal component analysis (PCA), which is the preferred approach for linear dimensionality reduction, may present serious limitations, in particular when samples are nonlinearly related, as often occurs in several two-dimensional electrophoresis (2-DE) datasets. An aggravating factor is that PCA robustness is impaired when the number of samples is small in comparison to the number of proteomic features, and this is the case in high-dimensional proteomic datas...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
The Use of Legendre and Zernike Moment Functions for the Comparison of 2-D PAGE Maps
The comparison of 2-D maps is not trivial, the main difficulties being the high complexity of the sample and the large experimental variability characterizing 2-D gel electrophoresis. The comparison of maps from control and treated samples is usually performed by specific software, providing the so-called spot volume dataset where each spot of a specific map is matched to its analogous in other maps, and they are described by their optical density, which is supposed to be related to the underlying protein amount. Here, a different approach is presented, based on the direct comparison of 2-D map images: each map is decompos...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Chemometric Multivariate Tools for Candidate Biomarker Identification: LDA, PLS-DA, SIMCA, Ranking-PCA
2-D gel electrophoresis usually provides complex maps characterized by a low reproducibility: this hampers the use of spot volume data for the identification of reliable biomarkers. Under these circumstances, effective and robust methods for the comparison and classification of 2-D maps are fundamental for the identification of an exhaustive panel of candidate biomarkers. Multivariate methods are the most suitable since they take into consideration the relationships between the variables, i.e., effects of synergy and antagonism between the spots. Here the most common multivariate methods used in spot volume datasets analys...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Multiple Testing and Pattern Recognition in 2-DE Proteomics
After separation through two-dimensional gel electrophoresis (2-DE), several hundreds of individual protein abundances can be quantified in a cell population or sample tissue. However, gel-based proteomics has the reputation of being a slow and cumbersome art. But art is not dead! While 2-DE may no longer be the tool of choice in high-throughput differential proteomics, it is still very effective to identify and quantify protein species caused by genetic variations, alternative splicing, and/or PTMs. This chapter reviews some typical statistical exploratory and confirmatory tools available and suggests case-specific guidel...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
2-DE Gel Analysis: The Spot Detection
The abundance of different proteins on a 2-DE gel is reflected by the shape, size, and intensity of the corresponding spots. Protein quantitation requires the conversion of an analog gel image into digital data, resulting into a catalog of individual spots listed as x, y positions, shape parameters, and quantitative values. So, it is possible to carry out objective comparisons of equivalent spots on different gels, determining whether a particular protein is more or less abundant in one sample compared with another. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Algorithms for Warping of 2-D PAGE Maps
Software-based image analysis of 2-D PAGE maps is an important step for the investigation of proteome. Warping algorithms, which are employed to register spots among gels, are able to overcome the difficulties due to the low reproducibility of this analytical technique. Over the years, the research of new matching and warping mathematical methods has allowed the development of several routine applications of easy-to-use software. This chapter describes common and basic spatial transformations used for the alignment of protein spots present in different gel maps; some recently new approaches are also presented. (Source: Spr...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
