High-sensitivity Analytical Approaches for the Structural Characterization of Glycoproteins

1.1. General Considerations Structural intricacies of carbohydrate molecules and their propensity to form varied linkages, substitutions, and branching patterns have fascinated many generations of chemists, as have the three-dimensional aspects of carbohydrate interactions with other biomolecules. The steadily increasing biochemical knowledge in this area has further added to the increasing importance of the field now referred to as “glycobiology” or, more generally, “glycoscience”. Yet, most of the emphasis over the last 50 years or so has been on two other classes of important biopolymers, namely nucleic acids and proteins. However, in the “post-genomic era”, complex carbohydrates can no longer be neglected, as it is becoming clear to many scientists that most mammalian proteins are glycosylated, and microbial systems and plants can have their own unique monosaccharide building blocks and special ways they can be interconnected and branched into unusual structures. Throughout evolution and the development of living organisms, glycoconjugates must have played major roles, no doubt due to their unusual biological selectivities, which, in turn, could well be due to the enormous information capacity of the “sugar code”.1,2 Throughout the 1980s, the multilateral importance of glycoconjugates in biology and medicine was recognized,3-6 albeit with an understanding that only new methodological approaches and systematic investigations would further define new vistas and provide intimate knowledge of how complex carbohydrates participate in all life processes. Today’s glycoscience is a multidisciplinary undertaking in which chemistry is expected to have an important role to describe the most complex structural aspects of sugars and their conjugates with other biological molecules. While the biological and biomedical relevance of studying glycosylation and sugar–protein and sugar–sugar interactions will undoubtedly be guided by advances in other respective fields (immunology, cancer research, parasitology, cell biology, and developmental biology, among others), the chemical disciplines’ two major tasks are to (a) isolate and structurally characterize biologically important glycoconjugates and (b) synthesize carbohydrate structures for biochemical investigations, enabling technologies and medical applications and providing new therapeutics. While the goals and directions of carbohydrate synthesis have been summarized elsewhere,7-11 the focus of our review has been on glycoanalytical chemistry. The synthetic and bioanalytical directions are not mutually exclusive, as new structural findings will undoubtedly provide further rationale for synthetic efforts and these, in turn, the availability of standards for structural verification. Since publication of the review on structural investigations of glycoconjugates at high sensitivity12 in these pages a decade ago, the field of analytical glycobiology has seen dramatic changes in its scope and depth. It is widely appreciated within the glycoscience community and increasingly by others that both new techniques and instrumentation and the established (albeit optimized) analytical methodologies have played very important roles in advancing the science of glycoconjugates to its current stage. Due to their different physical and chemical characteristics, the main classes of glycoconjugates, i.e. glycoproteins, glycolipids, polysaccharides, and proteoglycans with their highly charged constituents, glycosaminoglycans, demand somewhat specialized analytical and structural elucidation approaches. Our review will largely be focused on glycoproteins and their associated glycans, hoping that other scientists will describe the analytical aspects of the remaining glycoconjugate biomolecules elsewhere. The early advances in proteomics, the scientific area mostly preoccupied with identification and structural characterization of proteins, have led to diverse activities in protein post-transitional modifications (PTMs), which are often associated with important biological activities. Glycosylation of proteins is arguably the most widely spread and functionally most intriguing PTM in nature. It is already known that certain glycosylation patterns in proteins give rise to functional variance, with far-reaching consequences for health-disease issues, immunological disorders, toxicity effects, microbial invasion processes, etc. To investigate any of these highly important processes in sufficient molecular detail, analytical techniques capable of a high degree of structural elucidation and measurement sensitivity are currently needed. Within the plethora of new “-omics fields” (genomics, transcriptomics, lipidomics, metabolomics, etc.), the fields of glycoproteomics and glycomics have started to assume their respectable roles. Analytical glycobiology, representing both glycomics and glycoproteomics, now shares access to new measurement technologies that enable characterization and quantification of molecular processes in living organisms. Extensive glycomic and glycoproteomic data that can nowadays be generated with modern techniques and instrumentation are likely to enrich the “systems biology” approach.13-17 Both fields have started to contribute substantially to a better understanding of multicellular interactions in eukaryotic systems and important issues pertaining to human health and disease.18-23 Additionally, the long-held view that glycosylation is unimportant in prokaryotic systems is no longer defensible.24,25 Since our previous review12 in this journal, much progress has been achieved in terms of methodological developments toward better, more informative, and more sensitive measurements of glycoproteins and their glycan components. In addition, many conceptually important applications of new tools already point to the future needs for dealing with the enormous complexity of glycopeptides and oligosaccharide mixtures extracted from biological tissues and physiological fluids. The relatively recent interest of the pharmaceutical and biotech industries in recombinant glycoproteins, such as monoclonal antibodies, for treatment of cancer and other diseases,26-30 demands the use and further development of glycomic and glycoproteomic analytical procedures as well. Similarly to our previous report,12 the current review has been organized to discuss separately recent advances in glycoproteomics and glycomics, dealing first with the isolation and direct analysis of glycoproteins, followed by the description of advances in glycopeptide analysis and determination of the sites of glycosylation, and moving toward the analysis of complex glycan mixtures. Even more today than 10 years ago, mass spectrometry (MS) is the most prominent methodology in the arsenal of glycoprotein analysis tools. A number of new MS techniques, previously unexplored or insufficiently developed, are now at the center of attention of glycobiologists. At the sensitivity levels required by contemporary glycobiology, MS and tandem MS (MSn) techniques are currently the only means to provide reliable structural information. Carbohydrate derivatization (chemical modification of carbohydrates at microscale) uniquely enables certain MS measurements in terms of enhanced sensitivity and structural information. Due to the enormous “chemical space” for carbohydrate structural complexity,1,2 MS alone, no matter how sophisticated, is unlikely to provide all needed answers. However, in combinations with modern separation methodologies (different forms of chromatography and electrophoresis) that provide unique component resolution in time and space, MS detection and identification capabilities become enormously enriched. The past decade has seen substantial improvements in the chromatographic analysis of complex carbohydrates: (1) transition from the conventional-scale columns to capillary column dimensions, or even microchips, with the resulting gains in mass sensitivity of measurements; and (2) rapidly increasing use of stable and reliable hydrophilic column materials and graphitized carbon adsorbents. Further advances in capillary chromatographic separations pertain to effective resolution of very complex mixtures as well as the frequently needed separation of different isomers. Chromatographic advances of the recent years also relate to simple purifications of samples (analysis steps now often referred to as solid-phase extraction, or SPE) or the more sophisticated microcolumn lectin or affinity materials needed in group separations and preconcentration of certain glycoproteins for analysis. The past decade has also witnessed a rapid development of glycan array technologies, in which the surface-bound glycan structures (either synthesized or isolated from natural mixtures) are presented to glycan-binding proteins in biological samples.31-33 While these enabling technologies are novel and exciting, they will not be covered in this review, which primarily emphasizes techniques leading to structural elucidation of glycoproteins. Likewise, immunologically based measurements will not be discussed.