Description

Publisher details

Humana Press

Publications in this journal

• Methods for the Homology Modeling of Antibody Variable Regions

  Authors: Aroop Sircar

  Methods in molecular biology (Clifton, N.J.). 857:301.

  Antibodies are one of the critical molecules of our immune system and are unique in their enormous diversity required for recognizing various antigens. Antibodies are protein molecules and itsAntibodies are one of the critical molecules of our immune system and are unique in their enormous diversity required for recognizing various antigens. Antibodies are protein molecules and its antigen interacting region, the fragment variable (FV) is typically composed of a light (VL) and heavy (VH) chain. In particular three loops each at the tip of the VL and the VH, known as the complementarity determining region (CDR) loops, are responsible for binding to the antigen. While the framework regions of the VL and VH are relatively constant across the entire repertoire of antibodies, the conformation of the CDR loops vary extensively to enable the antibody to recognize different antigens. Three dimensional structures of antibodies illustrating the VL-VH relative orientation and the CDR conformations are needed to gain insight into antibody stability, immunogenicity and antibody-antigen interactions. Computational modeling provides a fast and inexpensive route for generating antibody structural models. This chapter highlights the various features crucial for creating a successful antibody homology model.

• A Taxonomist's View on Genomic Authentication.

  Authors: Bernard R Baum

  Methods in molecular biology (Clifton, N.J.). 862:1-12.

  A brief history of taxonomy, for the most part plant oriented, is provided, which demonstrates the use of morphology early on, through the stages when different technologies became available atA brief history of taxonomy, for the most part plant oriented, is provided, which demonstrates the use of morphology early on, through the stages when different technologies became available at different times until the present use of genomic tools. Genomic authentication facilitates with greater precision than ever before the identification of an organism or part thereof. In this chapter I made an attempt to stress that, in general, but more so for genomic authentication, the use of the variation inherent in taxa down to the lowest level of the hierarchy of classification needs to be used to achieve a high degree of correct authentication.

• Challenges in the DNA barcoding of plant material.

  Authors: Robyn S Cowan, Michael F Fay

  Methods in molecular biology (Clifton, N.J.). 862:23-33.

  DNA barcoding, using a short gene sequence from a standardized region of the genome, is a species identification tool which would not only aid species discovery but would also have applicationsDNA barcoding, using a short gene sequence from a standardized region of the genome, is a species identification tool which would not only aid species discovery but would also have applications ranging from large-scale biodiversity surveys through to identification of a single fragment of material in forensic contexts. To fulfill this vision a universal, relatively cheap, scalable system needs to be in place. The mitochondrial locus being used for many animal groups and algae is not suitable for use in land plants, and an appropriate alternative is needed.Progress has been made in the selection of two alternative regions for plant DNA barcoding. There are however many challenges in finding a solution that fulfills all the requirements of a successful, universally applicable barcode, and in the short term a pragmatic solution that achieves as much as possible and has payoffs in most areas has been chosen. Research continues in areas ranging from the technicalities of sequencing the regions to data analysis and the potential improvements that may result from the developing technology and data analysis systems.The ultimate success of DNA barcoding as a plant identification tool for all occasions depends on the building of a reference database and it fulfilling the requirements of potential users such that they are able to achieve valid results through its use, that would be more time consuming and costly, and less reliable using other techniques.

• DNA purification from multiple sources in plant research with homemade silica resins.

  Authors: Jian-Feng Li, Jen Sheen

  Methods in molecular biology (Clifton, N.J.). 862:53-9.

  DNA purification is a routine procedure in most plant laboratories. Although different kits are available in the market allowing convenient DNA purification, the cumulative cost of purchasingDNA purification is a routine procedure in most plant laboratories. Although different kits are available in the market allowing convenient DNA purification, the cumulative cost of purchasing multiple kits for a laboratory can be staggering. Here, we describe a protocol using homemade silicon dioxide matrix for DNA purification from Escherichia coli and Agrobacterium tumefaciens cells, PCR and restriction digestion mixtures, agarose gel slices and plant tissues. Compared with the commercial kits, this protocol enables easy DNA purification from diverse sources with comparable yield and purity at negligible expenses.

• Amplified fragment length polymorphism: an invaluable fingerprinting technique for genomic, transcriptomic, and epigenetic studies.

  Authors: Ovidiu Paun, Peter Schönswetter

  Methods in molecular biology (Clifton, N.J.). 862:75-87.

  Amplified fragment length polymorphism (AFLP) is a PCR-based technique that uses selective amplification of a subset of digested DNA fragments to generate and compare unique fingerprints for genomesAmplified fragment length polymorphism (AFLP) is a PCR-based technique that uses selective amplification of a subset of digested DNA fragments to generate and compare unique fingerprints for genomes of interest. The power of this method relies mainly in that it does not require prior information regarding the targeted genome, as well as in its high reproducibility and sensitivity for detecting polymorphism at the level of DNA sequence. Widely used for plant and microbial studies, AFLP is employed for a variety of applications, such as to assess genetic diversity within species or among closely related species, to infer population-level phylogenies and biogeographic patterns, to generate genetic maps, and to determine relatedness among cultivars. Variations of standard AFLP methodology have been also developed for targeting additional levels of diversity, such as transcriptomic variation and DNA methylation polymorphism.

• ISSR: A Reliable and Cost-Effective Technique for Detection of DNA Polymorphism.

  Authors: Maryam Sarwat

  Methods in molecular biology (Clifton, N.J.). 862:103-21.

  With the emergence of more and more molecular markers as useful tools in plethora of population genetic and phylogenetic studies, choice of marker system for a particular study has become mindWith the emergence of more and more molecular markers as useful tools in plethora of population genetic and phylogenetic studies, choice of marker system for a particular study has become mind boggling. These marker systems differ in their advantages and disadvantages, so it is imperative to keep in mind all the pros and cons of the technique while selecting one for the problem to be addressed.Here, we have shed light on the ISSR (intersimple sequence repeat) technique, as a marker of choice if one wants to go for properties such as reliability, simplicity, cost effectiveness, and speed, in addition to assessing genetic diversity between closely related individuals. We have outlined here the whole methodology of this technique with an example of Tribulus terrestris as case study.

• Authentication of Medicinal Plants by SNP-Based Multiplex PCR.

  Authors: Ok Ran Lee, Min-Kyeoung Kim, Deok-Chun Yang

  Methods in molecular biology (Clifton, N.J.). 862:135-47.

  Highly variable intergenic spacer and intron regions from nuclear and cytoplasmic DNA have been used for species identification. Noncoding internal transcribed spacers (ITSs) located in 18S-5.8S-26S,Highly variable intergenic spacer and intron regions from nuclear and cytoplasmic DNA have been used for species identification. Noncoding internal transcribed spacers (ITSs) located in 18S-5.8S-26S, and 5S ribosomal RNA genes (rDNAs) represent suitable region for medicinal plant authentication. Noncoding regions from two cytoplasmic DNA, chloroplast DNA (trnT-F intergenic spacer region), and mitochondrial DNA (fourth intron region of nad7 gene) are also successfully applied for the proper identification of medicinal plants. Single-nucleotide polymorphism (SNP) sites obtained from the amplification of intergenic spacer and intron regions are properly utilized for the verification of medicinal plants in species level using multiplex PCR. Multiplex PCR as a variant of PCR technique used to amplify more than two loci simultaneously.

• Loop-mediated isothermal amplification for the detection of plant pathogens.

  Authors: Lisa I Ward, Scott J Harper

  Methods in molecular biology (Clifton, N.J.). 862:161-70.

  Loop-mediated isothermal amplification (LAMP) is a technique involving the use of four to six primers (two inner primers, two outer primers, and two loop primers) and the strand displacement activityLoop-mediated isothermal amplification (LAMP) is a technique involving the use of four to six primers (two inner primers, two outer primers, and two loop primers) and the strand displacement activity of Bacillus subtilis-derived (Bst) DNA polymerase. The end result of strand displacement and loop formation and synthesis is the single-temperature amplification of a highly specific fragment from a DNA template at a much greater titre than that obtained with polymerase chain reaction. With LAMP, there are several methods to determine a positive reaction. Presented here are three alternative methods: gel electrophoresis, hydroxynaphthol blue colorimetric dye, and the fluorescent intercalating PicoGreen(®) reagent.

• Using GenBank® for Genomic Authentication: A Tutorial.

  Authors: James R Hennell, Paul M D'Agostino, Samiuela Lee, Cheang S Khoo, Nikolaus J Sucher

  Methods in molecular biology (Clifton, N.J.). 862:181-200.

  The GenBank(®) database is perhaps one of the most important repositories of genetic information. A researcher working in the field of genomic authentication must therefore be equipped with theThe GenBank(®) database is perhaps one of the most important repositories of genetic information. A researcher working in the field of genomic authentication must therefore be equipped with the skills needed to competently access the required information from this database whilst ultimately contributing their own data to it. This chapter presents a practical guide to using GenBank(®) to search for sequences, search and align unknown sequences using BLAST, and uploading and maintaining your own sequences. This chapter also details some other software helpful in sequence manipulation.

• Proteoglycans: gene cloning.

  Authors: Mauricio Cortes, James R Mensch, Miriam Domowicz, Nancy B Schwartz

  Methods in molecular biology (Clifton, N.J.). 836:3-21.

  Aggrecan is a large proteoglycan that plays roles in numerous tissues during vertebrate development and adult life. The 6,327-nt chick aggrecan coding sequence had been determined from overlappingAggrecan is a large proteoglycan that plays roles in numerous tissues during vertebrate development and adult life. The 6,327-nt chick aggrecan coding sequence had been determined from overlapping clones, but a full-length cDNA, needed for use in transgenic expression studies, had not been constructed. The strategy employed to do so was to generate two overlapping cDNA subfragments that shared a unique restriction site in the overlap and then join them at that site. These subfragments were obtained and cloned into the TOPO-TA vector pCR2.1. Digestion of the two constructs with the shared-site enzyme, XbaI, produced vector/5'-cDNA and 3'-cDNA fragments with XbaI-ends; these were ligated to produce the final full-length cDNA.

• Mapping of the Wnt/β-catenin/TCF response elements in the human versican promoter.

  Authors: Maziar Rahmani, Jon M Carthy, Bruce M McManus

  Methods in molecular biology (Clifton, N.J.). 836:35-52.

  Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and is considered to be crucial to several key cellular processes involved in development andVersican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and is considered to be crucial to several key cellular processes involved in development and disease. There is differential temporal and spatial expression of versican by multiple cell types and in different developmental and pathological timeframes. In order to fully appreciate the functional roles of versican as it relates to changing patterns of expression in development and disease, an in-depth knowledge of versican's biosynthetic processing is necessary. We have recently shown that β-catenin/T-cell factor (TCF) complex formation at the versican promoter site is essential for activation of versican transcription. The transcriptional activator β-catenin is the key mediator of the canonical Wnt signaling pathway. However, β-catenin does not itself bind DNA and thus functions via interaction with TCF/Lymphoid-enhancing factor (LEF) transcription factors. These proteins contain a high-mobility group (HMG) box that binds DNA in a sequence-specific manner. Thus, in the case of active Wnt signaling, β-catenin activates, in cooperation with proteins of the TCF/LEF family, the expression of a wide variety of target genes. The goal of this chapter is to describe the techniques used to elucidate the transcriptional control of versican by the β-catenin/TCF response elements in its promoter site and to demonstrate how this signaling may be assayed experimentally. These approaches provide insight into the transcriptional regulation of the versican gene and provide the basis for the identification of novel Wnt/β-catenin/TCF-regulated genes that are part of the signaling machinery regulating early embryogenesis, neoplasia, and cardiovascular remodeling.

• A novel strategy for a splice-variant selective gene ablation: the example of the versican V0/V2 knockout.

  Authors: María T Dours-Zimmermann, Dieter R Zimmermann

  Methods in molecular biology (Clifton, N.J.). 836:63-85.

  The complete knockout of genes that give rise to alternative splice products can often provide only an integral view of the dominant function(s) of all the isoforms they encode. If one of theseThe complete knockout of genes that give rise to alternative splice products can often provide only an integral view of the dominant function(s) of all the isoforms they encode. If one of these isoforms is indispensable for life, a constitutive and complete inactivation may even preclude any in vivo studies of later expressed splice-variants in mice. To explore function of the tissue-restricted versican V2 isoform during central nervous system maturation, for instance, we had to circumvent the early embryonic lethality of the complete knockout by employing a novel splice-variant-specific gene ablation approach. For this purpose, we introduced a preterm translational stop codon preceded by an ER-retention signal (KDEL) into the alternatively spliced exon 7 of the VCAN gene. This way the synthesis of the V2- and the V0-forms of the proteoglycan was entirely abolished in the mutant mice, most likely mediated by a KDEL-promoted intracellular degradation of the mutant fragment and by a nonsense-mediated decay mechanism. The expression of the vitally important V1-isoform and the smallest V3-variant remained, however, unaffected. Here we provide the details of our targeting strategy, the screening procedure, the generation of isoform-specific antibodies, and the transcript analysis and we supply the experimental protocols for the biochemical and immunohistological examinations of the mutant mouse strain Vcan(tm1.1Dzim).

• Glycosaminoglycan chain analysis and characterization (glycosylation/epimerization).

  Authors: Shuji Mizumoto, Kazuyuki Sugahara

  Methods in molecular biology (Clifton, N.J.). 836:99-115.

  Glycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate/heparin (HS/Hep) are linear polysaccharides and involved in the regulation of variousGlycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate/heparin (HS/Hep) are linear polysaccharides and involved in the regulation of various biological events through interaction with functional proteins. GAGs are modified by sulfation at various positions of each saccharide residue and the epimerization of uronic acid residues during the chain's biosynthesis, resulting in enormous structural diversity. This structural diversity is the basis for the wide range of biological activities of GAGs. Thus, the structural analysis of GAGs is key to understanding their biological functions. This chapter describes detailed instructions for the extraction and structural analysis of GAGs from cultured cells and tissues using a combination of GAG-degrading enzymes and high-performance liquid chromatography.

• Glycosaminoglycans: oligosaccharide analysis by liquid chromatography, capillary electrophoresis, and specific labeling.

  Authors: Derek J Langeslay, Christopher J Jones, Szabolcs Beni, Cynthia K Larive

  Methods in molecular biology (Clifton, N.J.). 836:131-44.

  Glycosaminoglycans (GAGs) are a class of biopolymers that include chondrotin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, heparin, and heparan sulfate. The GAGs are linearGlycosaminoglycans (GAGs) are a class of biopolymers that include chondrotin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, heparin, and heparan sulfate. The GAGs are linear polysaccharides that are microheterogeneous in composition and polydisperse in size. Because they have the most complex structures, this article is aimed at describing a step-by-step procedure for processing and analyzing heparin and heparan sulfate-derived oligosaccharides, although the basic protocols and procedures apply equally well to other members of the GAG family. The methods described in this manuscript include the preparation of oligosaccharides through enzymatic depolymerization, size fractionation by preparative scale size-exclusion chromatography (SEC), and disaccharide isomer analysis by reverse-phase ion-pair high-performance liquid chromatography (RPIP-HPLC) and capillary electrophoresis (CE).

• Use of neutrons reveals the dynamics of cell surface glycosaminoglycans.

  Authors: Marion Jasnin

  Methods in molecular biology (Clifton, N.J.). 836:161-9.

  Cell surface glycosaminoglycans (GAG), such as heparan sulfate (HS) and heparin, are key multifunctional cell regulators, which are involved in numerous molecular events associated with tumor growth,Cell surface glycosaminoglycans (GAG), such as heparan sulfate (HS) and heparin, are key multifunctional cell regulators, which are involved in numerous molecular events associated with tumor growth, metastasis, pathogen attachment, and immune response. GAG dynamically bind and regulate the activities of many signaling proteins such as growth factors, chemokines, and cytokines. GAG-binding interactions with proteins rely on the coupling between the geometry, flexibility, and rigidity of the polysaccharide chain. Understanding GAG dynamics at the molecular level can therefore provide fundamental insights into GAG function in a cellular context. Elastic incoherent neutron scattering is a powerful tool for the exploration of fast molecular motions in biological macromolecules. Recently, the technique was used to evaluate HS flexibility and rigidity on different timescales between the picosecond (ps) and the nanosecond (ns). Here, neutron spectroscopy experimental procedures are presented, with emphasis on the practical details necessary to prepare samples, run neutron scattering experiments, and extract the dynamics parameters from the data.

• In vivo scintigraphic imaging of proteoglycans.

  Authors: Elisabeth Miot-Noirault, Aurélien Vidal, Philippe Auzeloux, Caroline Peyrode, Jean-Claude Madelmont, Jean-Michel Chezal

  Methods in molecular biology (Clifton, N.J.). 836:183-98.

  In this chapter, we present the methods developed in our lab for the scintigraphic imaging and direct quantitative evaluation of proteoglycan (PG) distribution in vivo. These methods relate to (1)In this chapter, we present the methods developed in our lab for the scintigraphic imaging and direct quantitative evaluation of proteoglycan (PG) distribution in vivo. These methods relate to (1) the synthesis and radiolabeling of the NTP 15-5 with (99m)Tc, (2) preclinical scintigraphic imaging using laboratory animals, and (3) quantitative analysis of scintigraphic images.

• Analysis of aggrecan catabolism by immunoblotting and immunohistochemistry.

  Authors: Peter J Roughley, John S Mort

  Methods in molecular biology (Clifton, N.J.). 836:219-37.

  Aggrecan is essential for the normal function of articular cartilage and intervertebral disc, where it provides the ability for the tissues to withstand compressive loading. This property depends onAggrecan is essential for the normal function of articular cartilage and intervertebral disc, where it provides the ability for the tissues to withstand compressive loading. This property depends on both the high charge density endowed by its numerous chondroitin sulfate and keratan sulfate chains and its ability to form large molecular aggregates via interaction with hyaluronan. Degradation of aggrecan via the action of proteases takes place throughout life and the degradation products accumulate in the tissue and impair its function. Such degradation is exacerbated in degenerative or inflammatory joint disorders. The use of antibodies recognizing the various regions of aggrecan and the neoepitopes generated upon proteolytic cleavage has shown that matrix metalloproteinases and aggrecanases, members of the ADAMTS family, are responsible for aggrecan degradation, both throughout life and in disease. By using immunoblotting techniques, it is possible to determine the extent of aggrecan degradation and to identify the degradation products that have accumulated in the tissue, and immunohistochemistry allows the location of the aggrecan degradation to be established.

• Models for studies of proteoglycans in kidney pathophysiology.

  Authors: Scott J Harvey

  Methods in molecular biology (Clifton, N.J.). 836:259-84.

  Proteoglycans (PGs) impact many aspects of kidney health and disease. Models that permit genetic dissection of PG core protein and glycosaminoglycan (GAG) function have been instrumental toProteoglycans (PGs) impact many aspects of kidney health and disease. Models that permit genetic dissection of PG core protein and glycosaminoglycan (GAG) function have been instrumental to understanding their roles in the kidney. Matrix-associated PGs do not serve critical structural roles in the organ, nor do they contribute significantly to the glomerular barrier under normal conditions, but their abnormal expression influences fibrosis, inflammation, and progression of kidney disease. Most core proteins are dispensable for nephrogenesis (glypican-3 being an exception) and for maintenance of function in adult life, but their loss alters susceptibility to experimental kidney injury. In contrast, kidney development is exquisitely sensitive to GAG expression and fine structure as evidenced by the severe phenotypes of mutants for genes involved in GAG biosynthesis. This article reviews PG expression in normal kidney and the abnormalities caused by their disruption in mice and man.

• Shedding of cell membrane-bound proteoglycans.

  Authors: Eon Jeong Nam, Pyong Woo Park

  Methods in molecular biology (Clifton, N.J.). 836:291-305.

  Membrane-bound proteoglycans function primarily as coreceptors for many glycosaminoglycan (GAG)-binding ligands at the cell surface. The majority of membrane-bound proteoglycans can also function asMembrane-bound proteoglycans function primarily as coreceptors for many glycosaminoglycan (GAG)-binding ligands at the cell surface. The majority of membrane-bound proteoglycans can also function as soluble autocrine or paracrine effectors as their extracellular domains, replete with all GAG chains, are enzymatically cleaved and released from the cell surface by ectodomain shedding. In particular, the ectodomain shedding of syndecans, a major family of cell surface heparan sulfate proteoglycans, is an important posttranslational mechanism that modulates diverse pathophysiological processes. Syndecan shedding is a tightly controlled process that regulates the onset, progression, and resolution of various infectious and noninfectious inflammatory diseases. This review describes methods to induce and measure the shedding of cell membrane-bound proteoglycans, focusing on syndecan shedding as a prototypic example.

• Proteoglycans and osteolysis.

  Authors: Marc Baud'Huin, Céline Charrier, Gwenola Bougras, Régis Brion, Frédéric Lezot, Marc Padrines, Dominique Heymann

  Methods in molecular biology (Clifton, N.J.). 836:323-37.

  Osteolysis is a complex mechanism resulting from an exacerbated activity of osteoclasts associated or not with a dysregulation of osteoblast metabolism leading to bone loss. This bone defect is notOsteolysis is a complex mechanism resulting from an exacerbated activity of osteoclasts associated or not with a dysregulation of osteoblast metabolism leading to bone loss. This bone defect is not compensated by bone apposition or by apposition of bone matrix with poor mechanical quality. Osteolytic process is regulated by mechanical constraints, by polypeptides including cytokines and hormones, and by extracellular matrix components such as proteoglycans (PGs) and glycosaminoglycans (GAGs). Several studies revealed that GAGs may influence osteoclastogenesis, but data are very controversial: some studies showed a repressive effect of GAGs on osteoclastic differentiation, whereas others described a stimulatory effect. The controversy also affects osteoblasts which appear sometimes inhibited by polysaccharides and sometimes stimulated by these compounds. Furthermore, long-term treatment with heparin leads to the development of osteoporosis fueling the controversy. After a brief description of the principal osteoclastogenesis assays, the present chapter summarizes the main data published on the effect of PGs/GAGs on bone cells and their functional incidence on osteolysis.

• Gel-forming and cell-associated mucins: preparation for structural and functional studies.

  Authors: Julia R Davies, Claes Wickström, David J Thornton

  Methods in molecular biology (Clifton, N.J.). 842:27-47.

  Secreted and transmembrane mucins are important components of innate defence at the body's mucosal surfaces. The secreted mucins are large, polymeric glycoproteins, which are largely responsible forSecreted and transmembrane mucins are important components of innate defence at the body's mucosal surfaces. The secreted mucins are large, polymeric glycoproteins, which are largely responsible for the gel-like properties of mucus secretions. The cell-tethered mucins, however, are monomeric but are typically composed of two subunits, a larger extracellular subunit which is heavily glycosylated while the smaller more sparsely glycosylated subunit has a short extracellular region, a single-pass transmembrane domain, and a cytoplasmic tail. These two families of mucins represent high-molecular-weight glycoproteins containing serine and threonine-rich domains that are the attachment sites for large numbers of O-glycans. The high-M ( r ) and high sugar content have been exploited for the separation of mucins from the majority of components in mucus secretions. In this chapter, we describe current and well-established methods (caesium chloride density-gradient centrifugation, gel-filtration and anion-exchange chromatography, and agarose gel electrophoresis) for the extraction and purification of gel-forming and cell-surface mucins which can subsequently be used for a variety of structural and functional studies.

• Mass spectrometric analysis of mucin core proteins.

  Authors: Mehmet Kesimer, John K Sheehan

  Methods in molecular biology (Clifton, N.J.). 842:67-79.

  Mucins are difficult to handle for their identification and characterization via proteomic applications due to their heavily glycosylated nature (up to 90%), high molecular weight (200 kDa-200 MDa),Mucins are difficult to handle for their identification and characterization via proteomic applications due to their heavily glycosylated nature (up to 90%), high molecular weight (200 kDa-200 MDa), and size (Rg 10-300 nm). Their core proteins are extremely large and highly substituted with oligosaccharides, which only allow access to a highly restricted portion of their protein. For this reason, conventional 1D or 2D polyacrylamide gel-based proteomic approaches are not effective for identification and characterization of mucin molecules. In this chapter, we present our current protocol employing a modified shotgun proteomic approach to identify these complex glycoproteins.

• Analysis of assembly of secreted mucins.

  Authors: Malin E V Johansson, Gunnar C Hansson

  Methods in molecular biology (Clifton, N.J.). 842:109-21.

  Studies of assembly and secretion of gel-forming mucins are complex. The pulse-chase methods for mucins described here include metabolic radiolabeling and labeling in animals with azido-GalNAc. TheStudies of assembly and secretion of gel-forming mucins are complex. The pulse-chase methods for mucins described here include metabolic radiolabeling and labeling in animals with azido-GalNAc. The labeled mucins are analyzed by composite agarose-polyacrylamide gel electrophoresis and autoradiography or by mucus-preserving tissue fixation and Click-iT(®) chemistry.

• Glycomic work-flow for analysis of mucin o-linked oligosaccharides.

  Authors: Catherine A Hayes, Szilard Nemes, Samah Issa, Chunsheng Jin, Niclas G Karlsson

  Methods in molecular biology (Clifton, N.J.). 842:141-63.

  The high-throughput analysis of the glycosylation of high molecular weight proteins, such as mucins, has been the aim of glycomics initiatives for the last decade. Here, we present a work-flow forThe high-throughput analysis of the glycosylation of high molecular weight proteins, such as mucins, has been the aim of glycomics initiatives for the last decade. Here, we present a work-flow for the efficient and reproducible analysis of reduced oligosaccharides from a typical mucin sample. This work-flow can be applied to any similar samples of oligosaccharides. We include recently developed bioinformatic procedures for the statistical analysis of sample sets. These procedures can be applied in any laboratory environment, using free programs that are platform independent. The scripts are explained and can be adjusted to suit the individual experiment. Finally, a number of example results are given to highlight the use of the statistical analysis in a biological context.

• O-glycoproteomics: site-specific O-glycoprotein analysis by CID/ETD electrospray ionization tandem mass spectrometry and top-down glycoprotein sequencing by in-source decay MALDI mass spectrometry.

  Authors: Franz-Georg Hanisch

  Methods in molecular biology (Clifton, N.J.). 842:179-89.

  The sites of mucin-type O-glycosylation are difficult to predict, making structural analysis by mass spectrometry indispensible. This chapter refers to state-of-the-art techniques in the siteThe sites of mucin-type O-glycosylation are difficult to predict, making structural analysis by mass spectrometry indispensible. This chapter refers to state-of-the-art techniques in the site localization of O-linked glycans and their structural characterization in situ using tandem ESI and MALDI mass spectrometry. Detailed protocols are provided that describe the application of nano-LC-ESI-MS/MS with alternative fragmentation modes (collision-induced dissociation vs. electron-transfer dissociation) for the analysis of O-glycopeptides. Moreover, a top-down sequencing approach by MALDI-MS is presented that is based on the in-source decay of intact glycoproteins or large glycopeptides and allows a ladder sequencing of up to 70 amino acid residues from both termini with unequivocal assignment of modified sites.

Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.