The mucins (mucus glycoproteins) have long been a complex corner of glycoprotein biology. While dramatic advances in the separation, structural an- ysis, biosynthesis, and degradation have marked the progress in general glycop- tein understanding, the mucins have lagged behind. The reasons for this lack of progress have always been clear and are only now being resolved. The mucins are very large molecules; they are difficult to separate from other molecules present in mucosal secretions or membranes; they are often degraded owing to natural protective functions or to isolation methodology and their peptide and oligos- charide structures are varied and complex. Understanding these molecules has demanded progress in several major areas. Isolation techniques that protect the intact mucins and allow dissociation from other adsorbed but discrete molecules needed to be developed and accepted by all researchers in the field. Improved methods for the study of very large molecules with regard to their aggregation and polymerization were also needed. Structural analysis of the peptide domains and the multitude of oligosaccharide chains was required for smaller sample sizes, for multiple samples, and in shorter time. In view of these problems it is perhaps not surprising that the mucins have remained a dilemma, of obvious biological importance and interest, but very difficult to analyze.

This volume presents methods used for the analysis of glycoproteins at different levels—intact, subunit, glycopeptide, and monosaccharide--, and discusses and solves most analytical challenges that a scientist working on glycoproteins may come across. The chapters in this book cover topics such as the role of glycosylation on the properties of therapeutic glycoproteins; different analytical methods to characterize glycosylation, from the intact proteins to the glycan level, for both N-linked and O-linked glycoproteins; mass spectrometry imaging methodology for glycosylation analysis in tissues; approaches to characterizing glycosylation on cultured cells; and the use of cloud computing to deploy mass spectrometry data analysis. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Mass Spectrometry of Glycoproteins: Methods and Protocols is a valuable resource for scientists interested in learning more about this developing field.

This volume covers a wide spectrum of techniques and approaches that are used in the upstream and downstream processing for recombinant glycoprotein production. Chapters guide the reader through state-of-art of therapeutic recombinant glycoproteins, explores the patent literature, expression systems used for glycoproteins production, methods employed in the downstream processing of different glycoproteins, and information about analytical tools and formulation strategies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Recombinant Glycoprotein Production: Methods and Protocols aims to ensure successful results in the further study of this vital field

Glyco-engineering is being developed as a method to control the composition of carbohydrates and to enhance the pharmacological properties of monoclonal antibodies (mAbs) and other proteins. In Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols, experts in the field provide readers with production and characterization protocols of glycoproteins and glyco-engineered biopharmaceuticals with a focus on mAbs. The volume is divided in four complementary parts dealing with glyco-engineering of therapeutic proteins, glycoanalytics, glycoprotein complexes characterization, and PK/PD assays for therapeutic antibodies. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols serves as an ideal guide for scientists striving to push forward the exciting field of engineered biopharmaceuticals.

This comprehensive new resource in Springer s Methods in Molecular Biology series features contributions from leading researchers who provide expert advice and reproducible, cutting-edge protocols for examining glycoproteins through mass spectrometry."

This book focuses on recent developments of Pichia pastoris as a recombinant protein production system. Highlighted topics include a discussion on the use of fermentors to grow Pichia pastoris, information on the O- and N-linked glycosylation, methods for labeling Pichia pastoris expressed proteins for structural studies, and the introduction of mutations in Pichia pastoris genes by the methods of restriction enzyme-mediated integration (REMI). Each chapter presents cutting-edge and cornerstone protocols for utilizing P. pastoris as a model recomibinant protein production system. This volume fully updates and expands upon the first edition.

Conceived with the intention of providing an array of strategies and technologies currently in use for glyco-engineering distinct living organisms, this book contains a wide range of methods being developed to control the composition of carbohydrates and the properties of proteins through manipulations on the production host rather than in the protein itself. The first five sections deal with host-specific glyco-engineering and contain chapters that provide protocols for modifications of the glycosylation pathway in bacteria, yeast, insect, plants and mammalian cells, while the last two sections explore alternative approaches to host glyco-engineering and selected protocols for the analysis of the N-glycans and glyco-profiling by mass spectrometry. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls. Authoritative and extensive, Glyco-Engineering: Methods and Protocols offers vast options to help researchers to choose the expression system and approach that best suits their intended protein research or applications.