News
Detecting a sarinlike compound Because sarin, the chemical weapon released in the Tokyo subway almost three years ago, is so dangerous, its synthesis and distribution for basic research are tightly controlled, which is a major barrier to studying the pathophysiology of sarin poisoning. Masataka Nagao and colleagues at the University of Tokyo (Japan) described the synthesis of a sarinlike compound, bis(isopro-
pyl methyl)phosphonate (BIMP), and the development of a technique for diagnosing sarin poisoning. BIMP has the same phosphonate group and the same antiacetylcholinesterase activity as sarin. The IDr)(l and LI).-,, are 3.9 nM and 0.8 mg/kg, respectively. After exposing human reel blood cells to BIMP, BIMP-bound acetylcholinesterase was solubilized from erythrocyte membranes, purified by immunoaffinity chromatography, and digested with trypsin. The sarin hydrolysis products bound to acetylcholinesterase were released by alkaline phosphatase digestion, derivatized with trimethylsilyl(TMS) and measured bv GC/ MS Isopronvl methylphosDhonic and methvlphosnhonic •icifl^; w h i c h n m t h e ^'u*in hwlmfcnafi
Chromatogram of TMS derivatives of digested substances from BIMP-exposed red blood cells. 1 = isopropyl methylphosphonic acid, 2 = methylphosphonic acid. (Adapted with permission. Copyright Elsevier Science 1997.)
SCIENCE
An angle on DNA detection A detection method that doesn't require tags would be desirable for studying native DNA and DNA interactions in solution, which is where the nucleic acid's thermodynamics and structure most resemble their physiological state. Surface plasmon resonance (SPR) detection could be such a method. Robert M. Corn, Lloyd M. Smith, and colleagues at the University of WisconsinMadison described SPR imaging measurements of DNA hybridization at chemically modified gold surfaces {Anal. Chem. 1997 69 4939-47:4948-56). A surface plasmon is an electromagnetic wave that propagates along the interface between a metal and a dielectric. When the interface is illuminated with p-polarized light under conditions of total internal reflection at the "surface plasmon angle", the light can be coupled into surface plasmon modes, causing a measurable 92 A
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to the same solution." The imaging capability can be particularly useful in ruling out non-specific adsorption by comparing the responses of the correct DNA in one spot and incorrect DNA in another. In the present work, single-stranded DNA is attached to a modified gold surface via thiol-coupling surface chemistry. The immobilized layer serves as a probe that is exposed to a solution containing other molecules of interest. The SPR signal is magnified by exploiting the streptavidin-biotin interaction in a slightly unusual way. Rather than using the interaction to bind the DNA to the surface, they attach biotin to the free end of the DNA and "develop" the image by flowing streptavidin over the sample. "It's just a signal enhancement method that uses the bulk of the streptavidin molecule" Corn explains. The streptavidin is used only for monitoring oligonucleotides. When they detect PCR fragments, they can take advantage of the typically larger size of PCR products, which tend to be bulkier because of longer DNA sequences and attached primers. The sensitivity of the technique can also be enhanced by building DNA multilayers on the surface. However, SPR is not as adept at quantifying DNA as fluorescence detection, which is the most sensitive and popular DNA analytical method. "It's pretty clear from what we've done so far that fluorescence imaging is more sensitive and [easier than SPR]," says Smith. "We have to work hard to measure the hybridization of a PCR fragment by SPR, but it's pretty straightforward by fluorescence imaging. We're paying a price in out-of-the-box sensitivity. What do we get in return? The main thing we're going to get is applicability to systems in which it's not straightforward to attach a fluorescent tag." The true strength of the SPR technique, however, is in its ability to study interactions in solution, whether they be DNADNA DNA-protein, or protein-protein
decrease in the reflectivity. Because the angle at which SPR occurs depends upon the refractive index of the medium immediately adjacent to the metal, different materials and different amounts of the same material will cause different SPR responses. SPR can be performed in scanning mode, in which the sample is rotated relative to a fixed beam, or in imaging mode, in which a two-dimensional array detector is used to measure spatial variations in the reflected intensity. "The advantage of the imaging measurement," says Corn, "is that you can look at different parts of the surface and have multiple areas with different SPR images of hybridization and streptavidin adsorption onto two chemistries that probe oligonucleotides, P1 and P2. (a( Placement of the probe spots. can be simulta(b) SPR image following hybridization onto PI, (c) SPS image of the neously exposed same surface following regegeration ona hybridization ton2.
Analytical Chemistry News & &eatures, February 1, 1998
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 metiiodologies 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 Novotnv
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
