Protein extraction from body fluids is comparably straightforward, as these specimens already contain soluble glycoproteins. In the case of frozen or formalin-fixed tissues, more intensive physical disruption techniques such as pressure-assisted extraction can provide better yields. Established protocols using ultracentrifugation are available to enrich membrane glycoproteins but sample amount is a limiting factor. Following tissue homogenisation or sonication and cell-disruption, the extraction step facilitates access to the (glyco)proteins of interest, in particular as many membrane glycoproteins require the presence of detergents to ensure sufficient solubility. Methods for tissue or cell lysis and protein extraction are diverse, and often depend on the type and available amount of the biological sample. The first step of any glycoproteomics experiment is to access the glycoproteins of interest from the complex biological matrix. Strategies to identify glycoproteins and their glycosylation features from complex samples This mini-review intends to provide a concise introduction to the current strategies available to generate glycoproteomics data and to provide some guidance for designing tailored glycoproteomics experiments. While the literature harbours many excellent technical reviews covering specific aspects of glycomics (such as ) and glycoproteomics (examples include ) technologies and methodologies, there is a gap in the literature surveying the methods and practical issues of modern glycoproteomics relevant to beginners in the field. Understanding the molecular basis of how glycans are involved in health and disease requires technologies to precisely determine both the glycan structures (glycomics), and characterise their location and structure at discrete sites on glycoproteins (glycoproteomics) expressed by a cell or in an entire organ, body fluid, tissue or organism of interest. For a comprehensive introduction to the diverse biological functions of protein glycosylation, readers are referred to the freely available Essentials of Glycobiology textbook. This review aims to provide an ‘easy-to-digest’ introduction to the analytical approaches relevant for studying protein glycosylation.
Disease-associated changes in protein glycosylation are now considered a hallmark in many diseases, making glycans and glycoproteins promising molecular features with enormous diagnostic and prognostic value and potential therapeutic targets for precision medicine. Changes in cell glycosylation have been associated with systemic pathologies such as (but not limited to) inflammation, cancer or Alzheimer's disease. human ABO blood groups ), which, however, can influence the susceptibility to infectious diseases and create crucial population diversity. There are also examples of glycosylation ‘defects’ that do not impact normal development (e.g.
Congenital disorders of glycosylation (CDGs) are often embryonically lethal or phenotypically severe for affected individuals, emphasizing the essential role of glycosylation to life. In Eukaryotes, glycosylation is crucial for cell functions such as protein folding, regulating signalling or protein activity. This review introduces the common strategies to capture glycoprotein-specific and system-wide glycoproteome data from tissues, body fluids, or cells, and a perspective on how integration into a multi-omics workflow enables a deep identification and characterisation of glycoproteins - a class of biomolecules essential in regulating cell function. Examples of the variety of approaches, methodologies, and technologies currently used in the field are given. The review moves from the techniques that have been developed for the characterisation of single glycoproteins to technologies that may be used for a successful complex glycoproteome characterisation. This ‘Hitchhiker's guide to glycoproteomics’ is intended as a starting point for anyone who wants to explore the emerging world of glycoproteomics. Glycoproteomics, the study of glycans and their carrier proteins in a system-wide context, is becoming a powerful tool in glycobiology that enables the functional analysis of protein glycosylation. Changes in glycosylation are considered a hallmark of many diseases, thus making glycoproteins important diagnostic and prognostic biomarker candidates and therapeutic targets. Protein glycosylation is one of the most common post-translational modifications that are essential for cell function across all domains of life.
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