Dr. Orlando conducts research on using mass spectrometry (MS) to answer biological questions. He also is concerned with developing new methodologies to increase the amount of information obtained from MS experiments and to reduce the quantity of material needed for analysis. The procedures Dr. Orlando and his group have developed can currently elucidate the complete primary structures of the carbohydrate side chains of glycoproteins (including the stereochemistry, linkage, and anomeric configuration of each monosaccharide) from only low picomole quantities of sample.

The carbohydrate side chains of enzymatically glycosylated proteins play important, often essential, roles in the functions of glycoproteins. Carbohydrates linked through asparagine residues (N-linked) of glycoproteins participate in such health-related processes as hormone action, cancer, viral infection, and cell development and differentiation. The biological functions of carbohydrate chains attached through serine or threonine residues (O-linked) of glycoproteins are less well-defined, although these carbohydrate chains appear to be required for the biosynthesis, secretion, and compartmentalization of some glycoproteins. Alternatively, the non-enzymatic glycosylation (glycation) of proteins is believed to disrupt the normal structure and function of proteins and has been implicated in a range of health problems, particularly those associated with diabetes such as the development of cataracts.

The structural characterization of complex biologically active glycoproteins, essential to understanding their biological functions, currently holds numerous challenges for the biomedical researcher. Biomedically relevant glycoproteins typically can only be isolated in picomole quantities, while many of the techniques available for structurally analyzing the carbohydrate chains require at least nanomole quantities of material. Furthermore, no generally applicable strategy has been developed to determine O-linked glycosylation sites. The most widely used techniques for studying the carbohydrate portions of glycoproteins incorporate chemical or enzymatic release of the carbohydrate side chains from the peptide backbone prior to their structural analysis. However, the separation of the carbohydrate side chains from the peptide means that the point of attachment for each carbohydrate chain and the carbohydrate heterogeneity at each glycosylation site cannot be determined.

Dr. Orlando and his group are involved in several research projects that will continue their development of new MS strategies to structurally characterize glycoproteins. This work focuses on analyzing glycopeptides prior to removal of their carbohydrate side chains and reducing the sample quantities required for these MS procedures. Currently, they can characterize the complete primary structure of a glycoprotein from only 1-10 picomole of sample, approximately 5,000 times less material than is needed for present conventional methods. This work is also expected to produce general schemes for analyses that are particularly challenging for existing methodology, such as determining O-linked glycosylation and/or glycation sites. As new techniques are developed and refined, they are used to structurally characterize biologically significant glycated and glycosylated proteins.

For example, the discovery of elevated levels of glycated albumins and hemoglobins in diabetic patients has focused attention on the roles played by glycation in a range of health-related problems. Glycation is prevalent in diabetics in particular because of the frequent occurrence of high blood sugar levels in these patients. This modification of protein chains results from the irreversible addition of a saccharide to the free amino groups of lysines or the N-terminus of a protein. However, the lack of sensitive analytical procedures to structurally characterize glycated proteins has limited most research in this area to those glycated proteins that are easily obtained in large quantities, rather than to being able to study the effects of glycation in critical biomedical processes. Glycation of difficult-to-obtain proteins, therefore, may be responsible for a number of health-related problems in diabetics, including the development of cataracts. Glycation is believed to play a role in cataract development because it purportedly disrupts the structure of the eye lens proteins (crystallins). The tight, stable packing of the crystallins provides the optical characteristics necessary for vision. When this packing is disrupted by glycation, the refractive index of the lens is altered, causing light scattering and eventual lens opacity (cataracts).

Dr. Orlando's group is investigating the structural characterization of crystallins obtained from human eye lenses of healthy and diabetic patients. A major goal of this investigation is to determine the extent of crystallin glycation and the sites of sugar attachment in the crystallins of diabetic patients as compared to healthy individuals to learn more about the role of glycation in cataract development. The techniques developed during the study of glycated crystallins are expected to open up new areas of investigation concerning the role of glycation in other health-related problems associated with diabetes, such as kidney dysfunction, osteoporosis, and osteopenia. Dr. Orlando's work is supported by the National Institutes of Health, the National Science Foundation, and industrial sources.