Characterization of Nonribosomal Peptide Synthetases with NRPSsp
Bioinformatic sequence analysis allows the functional characterization of newly sequenced proteins. Nonribosomal peptide synthetases (NRPSs) are multi-modular enzymes involved in the biosynthesis of natural products. The current omics era has enabled the exponential growth of the sequenced NRPS, and it is important to characterize the final product of these synthetases. Here, how to achieve the prediction of substrates which bind to adenylation domains in NRPS with NRPSsp ( www.nrpssp.com ) bioinformatic tool is described. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Alignment-Free Methods for the Detection and Specificity Prediction of Adenylation Domains
Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10–40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST ...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics
Liquid chromatography–mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often>200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software s...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

The Use of ClusterMine36 for the Analysis of Polyketide and Nonribosomal Peptide Biosynthetic Pathways
Polyketides and nonribosomal peptides constitute two large families of microbial natural products. Over the past 20 years a broad range of microbial polyketide and nonribosomal peptide biosynthetic pathways have been characterized leading to a surfeit of genetic data on polyketide and nonribosomal peptide biosynthesis. We developed the ClusterMine360 database, which stores the antiSMASH-based annotation of gene clusters in the NCBI database, linking the structure of the natural product to the biosynthetic gene cluster. This database is searchable and enables the user to access multiple sequence files for phylogenetic analy...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Bioinformatics Tools for the Discovery of New Nonribosomal Peptides
This chapter helps in the use of bioinformatics tools relevant to the discovery of new nonribosomal peptides (NRPs) produced by microorganisms. The strategy described can be applied to draft or fully assembled genome sequences. It relies on the identification of the synthetase genes and the deciphering of the domain architecture of the nonribosomal peptide synthetases (NRPSs). In the next step, candidate peptides synthesized by these NRPSs are predicted in silico, considering the specificity of incorporated monomers together with their isomery. To assess their novelty, the two-dimensional structure of the peptides can be c...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Annotating and Interpreting Linear and Cyclic Peptide Tandem Mass Spectra
Nonribosomal peptides often possess pronounced bioactivity, and thus, they are often interesting hit compounds in natural product-based drug discovery programs. Their mass spectrometric characterization is difficult due to the predominant occurrence of non-proteinogenic monomers and, especially in the case of cyclic peptides, the complex fragmentation patterns observed. This makes nonribosomal peptide tandem mass spectra annotation challenging and time-consuming. To meet this challenge, software tools for this task have been developed. In this chapter, the workflow for using the software mMass for the annotation of experim...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

The Continuing Development of E. coli as a Heterologous Host for Complex Natural Product Biosynthesis
Heterologous biosynthesis of natural products is meant to enable access to the vast array of valuable properties associated with these compounds. Often motivated by limitations inherent in native production hosts, the heterologous biosynthetic process begins with a candidate host regarded as technically advanced relative to original producing organisms. Given this requirement, E. coli has been a top choice for heterologous biosynthesis attempts as associated recombinant tools emerged and continue to develop. However, success requires overcoming challenges associated with natural product formation, including complex biosynt...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro
The emergence of next-generation sequencing has provided new opportunities in the discovery of new nonribosomal peptides (NRPs) and NRP synthethases (NRPSs). However, there remain challenges for the characterization of these megasynthases. While genetic methods in native hosts are critical in elucidation of the function of fungal NRPS, in vitro assays of intact heterologously expressed proteins provide deeper mechanistic insights in NRPS enzymology. Our previous work in the study of NRPS takes advantage of Saccharomyces cerevisiae strain BJ5464-npgA as a robust and versatile platform for characterization of fungal NRPSs. H...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Facile Synthetic Access to Glycopeptide Antibiotic Precursor Peptides for the Investigation of Cytochrome P45 Action in Glycopeptide Antibiotic Biosynthesis
The glycopeptide antibiotics are an important class of complex, medically relevant peptide natural products. Given that the production of such compounds all stems from in vivo biosynthesis, understanding the mechanisms of the natural assembly system—consisting of a nonribosomal-peptide synthetase machinery (NRPS) and further modifying enzymes—is vital. In order to address the later steps of peptide biosynthesis, which are catalyzed by Cytochrome P450s that interact with the peptide-producing nonribosomal peptide synthetase, peptide substrates are required: these peptides must also be in a form that can be conju...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases
Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes consisting of catalytic domains. The substrate specificities of adenylation (A) domains determine the amino-acid building blocks to be incorporated during nonribosomal peptide biosynthesis. The A-domains mediate ATP-dependent activation of amino-acid substrates as aminoacyl-O-AMP with pyrophosphate (PPi) release. Traditionally, the enzymatic activity of the A-domains has been measured by radioactive ATP–[32P]-PPi exchange assays with the detection of 32P-labeled ATP. Recently, we developed a colorimetric assay for the direct detection of PPi as a ye...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains
A series of inhibitors have been designed based on 5′-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of l-Phe-AMS (l-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert l-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Secondary Metabolic Pathway-Targeted Metabolomics
This chapter provides step-by-step methods for building secondary metabolic pathway-targeted molecular networks to assess microbial natural product biosynthesis at a systems level and to aid in downstream natural product discovery efforts. Methods described include high-resolution mass spectrometry (HRMS)-based comparative metabolomics, pathway-targeted tandem MS (MS/MS) molecular networking, and isotopic labeling for the elucidation of natural products encoded by orphan biosynthetic pathways. The metabolomics network workflow covers the following six points: (1) method development, (2) bacterial culture growth and organic...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay
We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news

In Situ Analysis of Bacterial Lipopeptide Antibiotics by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a technique developed in the late 1990s enabling the two-dimensional mapping of a broad variety of biomolecules present at the surface of a sample. In many applications including pharmaceutical studies or biomarker discovery, the distribution of proteins, lipids or drugs, and metabolites may be visualized within tissue sections. More recently, MALDI MSI has become increasingly applied in microbiology where the versatility of the technique is perfectly suited to monitor the metabolic dynamics of bacterial colonies. The work described here i...
Source: Springer protocols feed by Protein Science - February 6, 2016 Category: Biochemistry Source Type: news

The Assembly Line Enzymology of Polyketide Biosynthesis
Polyketides are a structurally and functionally diverse family of bioactive natural products that have found widespread application as pharmaceuticals, agrochemicals, and veterinary medicines. In bacteria complex polyketides are biosynthesized by giant multifunctional megaenzymes, termed modular polyketide synthases (PKSs), which construct their products in a highly coordinated assembly line-like fashion from a pool of simple precursor substrates. Not only is the multifaceted enzymology of PKSs a fascinating target for study, but it also presents considerable opportunities for the reengineering of these systems affording a...
Source: Springer protocols feed by Protein Science - February 6, 2016 Category: Biochemistry Source Type: news

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 ). Variou...
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

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

Spot Matching of 2-DE Images Using Distance, Intensity, and Pattern Information
We describe a simple and accurate method which allows to automatically and accurately match spots in 2-DE images. The method consists of simultaneously exploiting the distance between the spots, their intensity, and the pattern formed by their spatial configuration. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news

Image Pretreatment Tools II: Normalization Techniques for 2-DE and 2-D DIGE
Gel electrophoresis is usually applied to identify different protein expression profiles in biological samples (e.g., control vs. pathological, control vs. treated). Information about the effect to be investigated (a pathology, a drug, a ripening effect, etc.) is however generally confounded with experimental variability that is quite large in 2-DE and may arise from small variations in the sample preparation, reagents, sample loading, electrophoretic conditions, staining and image acquisition. Obtaining valid quantitative estimates of protein abundances in each map, before the differential analysis, is therefore fund...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news

Image Pretreatment Tools I: Algorithms for Map Denoising and Background Subtraction Methods
One of the critical steps in two-dimensional electrophoresis (2-DE) image pre-processing is the denoising, that might aggressively affect either spot detection or pixel-based methods. The Median Modified Wiener Filter (MMWF), a new nonlinear adaptive spatial filter, resulted to be a good denoising approach to use in practice with 2-DE. MMWF is suitable for global denoising, and contemporary for the removal of spikes and Gaussian noise, being its best setting invariant on the type of noise. The second critical step rises because of the fact that 2-DE gel images may contain high levels of background, generated...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news

Comparative Evaluation of Software Features and Performances
Analysis of two-dimensional gel images is a crucial step for the determination of changes in the protein expression, but at present, it still represents one of the bottlenecks in 2-DE studies. Over the years, different commercial and academic software packages have been developed for the analysis of 2-DE images. Each of these shows different advantageous characteristics in terms of quality of analysis. In this chapter, the characteristics of the different commercial software packages are compared in order to evaluate their main features and performances. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news

A Novel Gaussian Extrapolation Approach for 2-D Gel Electrophoresis Saturated Protein Spots
Analysis of images obtained from two-dimensional gel electrophoresis (2-D GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software currently available have proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this chapter, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconst...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news