Structural analysis of glycoproteins on a MALDI plate One of the biggest problems in the structural determination of glycoproteins has been the lack of sensitive methods. Recent advances in MALDI (matrix-assisted laser desorption/ionization) MS, however, are changing all of that. In this issue of Analytical Chemistry, Milos V. Novotny and Yehia Mechref of Indiana University report a new method for the structural analysis of N-linked oligosaccharides derived from low-microgram and submicrogram quantities of purified glycoproteins (p. 455) The entire procedure is carried out on a MALDI plate starting with the cleavage of the sugar from the polypeptide backbone The procedure is much faster than conventional methods and can be completed in one day or less
including carbohydrate sequencing and determination of sugar linkage forms, has traditionally involved tedious, multistep processes that often require milligram to gram quantities of sample. "We have been trying to develop more sensitive methodologies for determining the structure of glycans for a number of years. In some of our earlier approaches we used capillary electrophoresis and laser-induced fluorescence, which gave us a lot of sensitivity and resolving power for glycan mixtures, but it did not tell us what the compounds were," says Novotny. "Steadily, advances in MS have been made. We contributed to some of them by developing a MALDI matrix that works remarkably well for sugars." This new arabinosazone matrix has been used to study the fragmentation of sugars, leading to enzyme-based sequencing and linkage determination It does not perturb chemical structures and it can be used for a number of different things says Novotny
Glycoproteins are important in a wide range of biochemical processes, from egg fertilization to the targeting of aging cells. They have extremely complex structures, with multiple substitutions at various glycosylation sites. Glycoproteins contain covalently bound oligosaccharides, most with branched structures. Complete structural analysis of oligosaccharides, also referred to as "glycans",
Extensive sample manipulation commonly leads to loss of material and, ultimately, inaccurate results. "Normally, sample manipulation is a big problem," remarks Novotny. "When you start with small quantities of material, it is essential that you do not lose any before you get to the final [MS] determination." Complete processing of a glycoprotein on a MALDI plate thus helps prevent sample losses.
interactions. "There's a dearth of ways of looking at [protein-protein interactions]," says Smith. "SPR could be a powerful way of taking complex mixtures, picking out molecules of interest, and seeing what [component] in some mixture binds to those molecules. You can imagine putting a thousand proteins on the surface, fishing into an extract that contains a thousand proteins, and seeing which ones bind to which. Then you'd be able to map the protein-protein interactions that are occurring in vivo under natural conditions." A potential application of this technique is in DNA diagnostics. "There's an emerging market for detecting sequence variations," says Smith. "One approach is hybridization to surfaces. SPR is a technique that could be used quite inexpensively. If the sensitivity could go up by an order of magnitude, it could be a competitive technique for looking at hybridization-based discrimination of mutations." Celia Henry
Electrospray, but not electrospray Electrospray is well-known as an ionization technique for biomolecules, but Murray Hackett of the University of Washington's Department of Medicinal Chemistry didn't have much luck using negative ion electrospray for glycolipids. Hackett and Houle Wang solved their problem by developing a cousin to the standard electrospray interface—a cylindrical capacitor interface for electrospray ionization (Anal. Chem. 1998,70,205-12). With conventional electrospray, Hackett had trouble mainly with anion analysis at nanoliter per minute flow rates. "For various reasons we don't get the sensitivity [with anions]. There's a hypothesis that many anions simply lose the charge, that you may make the ion transiently, but it doesn't hold on to the negative charge long enough to see it in the mass analyzer."
When mixtures are not complicated, a separation step is not required for the structural analysis of glycans, which serve as a fingerprint of the glycoprotein. "In the less complicated glycoproteins, we see basically four peaks or a limited number of parent peaks. You have to keep track of the peak differences in mass, but it does work in many cases. In some instances, however, the mixtures are too complex and a separation step will be necessary," explains Novotny. The total structural characterization of N-linked oligosaccharides was determined for several glycoproteins, including ribonuclease B, human a,-acid glycoprotein, bovine fetuin, diamino oxidase from porcine kidney, and ovalbumin by the on-plate procedure. The approach has the potential to be used for analysis of some of the most challenging glycoproteins, such as receptor proteins at biological membranes. In addition to N-linked oligosaccharides, glycoproteins also contain O-glycans. Novotny and co-workers are currently developing a parallel method for the structural determination of O-linked oligosaccharides. 'We are not quite down to die sensitivity we need, but we have already developed a chemical procedure for the cleaving of O-glycans," says Novotny. Britt Erickson
The capacitor interface differs from a standard electrospray interface in several vays. There is no external high-voltage electric field; instead, the high-voltage electrode
A schematic of the cylindrical capacitor electrospray interface.
Analytical Chemistry News & Features, February 1, 1998 9 3 A
