News
NEWS FROM THE EASTERN ANALYTICAL SYMPOSIUM Elizabeth Zubritsky reportsfromSomerset, NJ
Chemometrics give NMR a boost These days, NMR is working smarter, not harder. Using chemometric analysis of *H NMR spectra, Elaine Holmes and colleagues at Imperial College (U.K.) have shown that they can detect differences between two strains of rats and that they can identify tissue-specific toxins by analyzing the animals' urine samples. These are the newest parts of a two-year-old effort to construct a database of known toxins to better understand their mechanisms of action and to help with the screening of new compounds. Data on at least 50 toxins have been collected thus far, based on analyses of urine samples taken over seven days from treated rats. (A second database based on blood plasma samples also has been started.) The scientists collected NMR spectra at 400 MHz on the sample and divided an approximately 10-ppm section of interest into 0.04-ppm parts. After integrating the regions and compensating for concentration differences, they used chemometric analyses. First, unsupervised techniques such as principal component analysis (PCA) were used to group the data and if a grouping was found supervised techniques such cis SIMCA (soft independent modeling of class analogy) were used to optimize the group separation One advantage of chemometrics is that it can pick out details "you won't necessarily notice by eye", Holmes says. "Ifs a good way of detecting subtle changes." The unsupervised clustering analysis, for example, recently revealed differences between the SpragueDawley strain of rats and Han Wistar rats. The analysis also clearly showed that these differences are much smaller than those between toxin-treated and control animals. That kind of information can be used to define the range of normal physiological variance and to prescreen animals for experiments Holmes says. Similarly, SIMCA analysis made it possible to tease out indicators of tissuespecific toxicity from the complex spectra of the samples, Holmes says, revealing the "combination of markers that fingerprint the toxicity type". To date, the researchers have correctly classified nearly all of the muscle-, glomerular-, and renalcortical-specific toxins they have tested, she says. They have also detected signs 88 A
Coomans plot showing the clustering of normal Han Wistar rats (red), normal Sprague-Dawley rats (yellow), and toxin-treated animals (blue).
of recovery in the plasma of hemodialysis patients one day earlier than the standard clinical tests. The goal, Holmes says, is to develop expert systems that can be usedtoscreen new compounds, to determine how accurately animals model human disease, and, eventually, to reveal how well a patient's treatment is working. The research is just getting started, she says, but ifs beginning to show possibilities.
Epitope mapping MS has been used for several years to map epitopes—the actual portions of the protein that are recognized by antibodies. Thus far, these epitopes have been linear, meaning that the residues that make up the epitope lie next to each other in sequence. The alternative is a discontinuous epitope in which the residues are not sequential but are brought together topologically by the folding of the protein. Now, Kenneth Tomer and colleagues at the National Institute of Environmental Health Sciences have reported an approach using MS that should be applicable to discontinuous epitopes as well.
Using a MALDI-TOFMS technique in which antibody-coated beads are crystallized with the matrix, the researchers mapped the binding epitope for the large, heavily glycosylated HIV envelope protein gpl20. For binding, the scientists used a polyclonal antibody—which is produced by many different antigen-sensitive cells and whose epitopes, therefore, vary slightly in specificity and affinity. The researchers used two approaches for mapping. They allowed the protein to bind to the antibody and then digested the unprotected parts of the protein a technique known as epitope excision or proteolytic footprinting. They also digested the protein and then allowed the fragments to bind to the antibody—a technique known as epitope extraction Bv using consecutive proteolytic reactions the fine structures of the epitopes were manned The results showed that the major epitope recognized by the antibody consisted of residues 472-478, the sequence RRWQRE (where the letters represent the amino acids). Minor epitopes were contained within the sequences APTKAKRRWQ, APTKAKRRV, APTKAKRR and RRWQR Because the researchers were able to characterize several epitopes simultaneously, they say that the technique should be valid for discontinuous epitopes. They add that combining such experiments with secondary and tertiary structural information from databases and molecular models should make identification unambiguous. The experiment was also important, Tomer says, because gpl20 is a large (Mr -100,000), glycosylated protein, and epitope mapping on such proteins has not been done before. "One of the fascinating parts of this technique... is the ability to work on a large protein in its native conformation," he says.
Coming in March: Made to Measure A special publication entitled "Made to Measure" will be mailed with the March 1 issue of Analytical Chemistry and Analytical Chemistry News & Features. Written ena produced by the staffs of Analytical Chemistry and Today'' Chemist at Work in recognition of Pittcon's 50th anniversary, this publication will include a 50-year history of the analytical instrument business, a look at how federal agencies such as EPA and FDA have influenced the development of analytical instruments, predictions for the future of the business, a preview of the old instruments exhibit to be presented at Pittcon this year, biographies of the leaders of the analytical instrument business as chosen by the organizers of Pittcon, and a large poster summarizing the history of the analytical instrument enterprise. The issue will also include many old photographs and instrument ads. Copies of "Made to Measure" will also be distributed at Pittcon.
Analytical Chemistry News & Features, February 1, 1999
