Current Proteomics - Volume 8, Issue 4, 2011

Glycomics and glycoproteomics continue to be highly dynamic and interesting research areas due to the needs to comprehensively understand the biological attributes of glycosylation in many important biological functions such as immune response, cell development, cellular differentiation/adhesion, and host-pathogen interactions. Moreover, aberrant glycosylation for decades has been recognized as the attribute of many mammalian diseases, including osteoarthritis, cystic fibrosis and cancer. This being said, glycomic and glycoproteomic analyses remain to be analytically very challenging mainly due to the high complexity and microheterogeneity of glycosylation processes and machineries. Therefore, more cutting-edge-analytical approaches are needed to allow more comprehensive characterization of glycosylation and subsequently better understanding of its biological roles. The ultimate goal of the research activities in glycomics and glcyoproteomics is the comprehensive characterization of glycoprotein glycosylation sites and microheterogeneity of such sites. Accordingly, several glycomic and glycoproteomic approaches are continuously being refined and developed to facilitate the achievement of this ultimate goal. Since interpretation and processing of glycomic and glycoproteomic data are laborious and time consuming, the development of different bioinformatic tools aiding rapid and automated data interpretation and processing are also immensely needed. The major focus of this issue is to review the most recent progress made in employing mass spectrometry in the fields of glycomics and glycoproteomics. The article of Loubna A. Hammad and Yehia Mechref reviews the utility of mass spectrometry for the quantitative determination of monosaccharides associated with glycoconjugates, while that of Carol L. Nilsson reviews the progress in utilizing lectin techniques in glycoproteomics. Kathryn R. Rebecchi, Carrie L. Woodin, and Heather Desaire review the recent advances in quantitative glycoproteomics, while recent advances in the structural characterization of carbohydrates by Fourier transform tandem mass spectrometry are reviewed by Wen Zhou and Kristina Hakansson. Advances in the use of mass spectrometry for sulfoglycans and glycosaminoglycans are detailed and described by Karlsson et al. and Zaia et al. respectively. Last but not least, the new advances in the development of the much needed bioinformatic tools to aid in handling and interpreting glycomic and glycoproteomic data are authoritatively reviewed by Anoop M. Mayampurath, Chuan-Yih Yu, Yehia Mechref, and Haixu Tang. We believe this hot topic issue will provide a wealth of information to those interested in glycomic and glycoproteomic analyses which are research areas currently filled with excitements and fulfilling enjoyments. We also believe this issue will serve as a reference for those indulged in this very interesting and stimulating area of research known as glycobiology.

One of the greatest challenges of the post-genomic and post-proteomic era is to understand the role of posttranslational modifications in global biological systems. Protein glycosylation is one of the most frequent posttranslational modifications but has been poorly studied in global proteomes because of analytical challenges associated with glycan structural characterization. In recent years, technical advances in the study of protein glycosylation have been achieved by the use of lectins as an enrichment tool for subproteomes modified by specific types of glycan structures. Many clinical biomarkers are glycoproteins and glycoproteomic studies that use lectin affinity techniques have been shown to have a superior ability to determine structure-specific biomarkers of disease. This article reviews key techniques and workflows, including the use of single- and multiple lectin columns and glycan detection by lectins.

Effective and accurate analysis of free monosaccharides and monosaccharides bound to glycoconjugates, including glycoproteins and glycolipids have been achieved through the development of different analytical methods. Although all of these analytical methods appears to be applicable to the quantification of monosaccharides, quantification through coupling of liquid chromatography to mass spectrometry (LC-MS) is currently considered to be one the most effective analytical methods. This review focuses on the development of LC-MS methods for the analysis of monosaccharides, and evaluates the different analytical approaches including various ionization and detection techniques that have been used in order to analyze and quantify monosaccharides using mass spectrometry.

Glycoproteomics is an emerging field focused on analysis of glycosylated proteins. Glycoproteins, especially N-linked glycoproteins, have been shown to be increasingly important in biomarker analyses and biopharmaceutical drug development. Thus, relative quantitation of glycoproteins by mass spectrometry is surfacing as a powerful tool. Examples of quantitative glycoproteomic analyses include monitoring changes in protein expression levels, glycosylation site occupancy, and site-specific glycosylation. Specific quantitative strategies can be varied; however, most glycoproteomics investigations involve a glycoprotein/glycopeptide enrichment step, which is important for separating glycoproteins/ glycopeptides from the rest of a sample matrix. Depending on the goal of the quantitative study, after enrichment, the glycans can either be deglycosylated, or released, from the protein for analysis at the peptide level or analyzed as glycopeptides. Deglycosylation is typically performed in studies involving protein expression level changes or monitoring glycosylation site occupancy; whereas quantitative glycopeptide analysis is critical for monitoring changes in glycosylation in a protein-specific or glycosylation site-specific manner. Quantitation can be performed through a label-free or labeling approach. This review focuses on contrasting the various approaches' strengths and weaknesses and providing example applications of these state-of-the-art methods.

The research area of glycomics is maturing and it is realized that it is as difficult to identify the relevance of glycosylation amongst biological house-keeping as it is with any of the other omics techniques. This review focuses on sulfoglycomics of N-linked and O-linked oligosaccharides to identify relevant biological glycomics questions. The review describes how sulfated oligosaccharides have been analyzed in the past, with the focus on MS and its current application using ESI and MALDI. Also included are the particular issues of sample preparation of sulfated oligosaccharides, including enrichment and separation using capillary and nano liquid chromatography. Structural characterization of sulfated oligosaccharides is currently carried out by collision induced dissociation, but recent developments focus on increasing the structural information by improved derivatization techniques followed by MS analysis and fragmentation of derivatized intact sulfooligosaccharides. Significant progress has been shown in this field in the application of permethylation derivatization. Current trends for fragmentation of biomolecules using novel techniques are also discussed with emphasis on sulfoglycomics. The conclusion of the review is that in order to understand the largely unexplored area of sulfation of oligosaccharides, there are techniques that need to be adapted and optimized to address the structural characterization, ranging all the way from appropriate sample preparation to effective MS analysis.

Fourier transform tandem mass spectrometry (MS/MS) provides high mass accuracy, high sensitivity, and analytical versatility and has therefore emerged as an indispensable tool for structural elucidation of biomolecules. Glycosylation is one of the most common posttranslational modifications, occurring in ∼50% of proteins. However, due to the structural diversity of carbohydrates, arising from non-template driven biosynthesis, achievement of detailed structural insight is highly challenging. This review briefly discusses carbohydrate sample preparation and ionization methods, and highlights recent developments in alternative high-resolution MS/MS strategies, including infrared multiphoton dissociation (IRMPD), electron capture dissociation (ECD), and electron detachment dissociation (EDD), for carbohydrates with a focus on glycans and proteoglycans from mammalian glycoproteins.

Glycans play multiple functional roles in living cells, from acting as energy sources to mediating intracellular signaling and cell-cell communications. Glycosylation is a common post-translational modification (PTM), known to occur in over 50% of human proteins. Understanding the structure of glycans, the nature and functions of protein glycosylation along with studying interactions between glycans and glycan-binding proteins are crucial developing indicators for disease and the study of pathogenic infections. The use of bioinformatic approaches and computational methods aids in reaching this goal. This paper aims to review some of the bioinformatics techniques and tools that are specifically developed and utilized for studying glycans and glycosylated proteins. While significant progress has been made, bioinformatics tools available for glycobiology are still not comparable to those developed for proteomics and genomics. On the other hand, glycans are ideal models for systems biology studies and it is expected that bioinformatics methods that encompass a systems approach will be very fruitful.

The glycosaminoglycans (GAGs) are linear polysaccharides expressed on animal cell surfaces and in extracellular matrices. Their biosynthesis is under complex control and confers a domain structure that is essential to their ability to bind to protein partners. Key to understanding the functions of GAGs are methods to determine accurately and rapidly patterns of sulfation, acetylation and uronic acid epimerization that correlate with protein binding or other biological activities. Mass spectrometry (MS) is particularly suitable for the analysis of GAGs for biomedical purposes. Using modern ionization techniques it is possible to accurately determine molecular weights of GAG oligosaccharides and their distributions within a mixture. Methods for direct interfacing with liquid chromatography have been developed to permit on-line mass spectrometric analysis of GAGs. New tandem mass spectrometric methods for fine structure determination of GAGs are emerging. This review summarizes MS-based approaches for analysis of GAGs, including tissue extraction and chromatographic methods compatible with LC/MS and tandem MS.