DON FREDERICK
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Leigh Francis of the Australian Wine Research Institute assesses wine quality the old-fashioned way.
tridge into a test tube. Sulfuric acid is added, and the test tube is heated to break down the glycosides. The researchers quantify the resulting sugar level by using conventional enzymatic spectrophotometric analysis with a commercially available kit and automated analyzer. The procedure is a little too complicated for wine makers to do themselves, and the hard-pressed institute staff has only been able to complete about 10 G–G assays a day. But help is on the way from Advanced Rapid Robotic Manufacturing, a firm near Adelaide, which is perfecting an automated system that could potentially turn out 100 assays a day. According to Francis, the fully automated system will homogenize grape samples and perform an
extraction and a centrifuging step, solid-phase extraction, and hydrolysis on 96 samples simultaneously. “We’re still building the system, and it may be another six months before it is finished,” comments Francis. As an alternative to the assay, the scientists are testing near-IR spectroscopy for determining G–G levels in grapes. If the technology can be incorporated into a relatively inexpensive, hand-held instrument, wine makers and grape growers could assess the flavor potential of fruit on the vine or at the weigh-bridge instead of waiting for costly and time-consuming reports. Aware that the glycosides have a major impact on wine flavor, researchers are now trying to zero in on the specific compounds that please the palate. For instance, betaionone, which gives some pinot noirs a powerful punch, also appears in raspberries. Is reisling a reminder of the days of wine and roses? It wouldn’t be surprising. The compounds linalool and geraniol, which are found in the reisling grape, also appear in roses. In the end, the researchers have every reason to smell the roses, Francis says. They are working on a GC/MS method, which uses deuterium-labeled analogues of the aroma compounds as internal standards, to quantify the flavor compounds on a routine, relatively rapid basis. He adds that several standards have been synthesized and that the researchers are at the point of developing standard curves and validating the analyses. “In a reasonably short time, possibly two years,” he predicts, “we will have an analytical method for the determination of key compounds derived from grapes.” Don Frederick
2-D PAGE gets competition Anyone who has separated proteins using 2-D polyacrylamide gel electrophoresis (PAGE) knows how labor-intensive and cumbersome it is. But despite rapid growth in the field of proteomics, there aren’t many other options for analyzing whole-cell protein expression. That, however, could be about to change. In the March 15 issue of Analytical Chemistry (pp 1099–1111), David Lubman and coworkers at the University of Michigan describe a new twodimensional (2-D) liquid-phase separation method capable of resolving large numbers of cellular proteins and generating quantitative maps of protein expression. In the first dimension, proteins are separated according to pI using a commercially available liquid-phase isoelectric focusing (IEF) separation device. The separation takes about 3–5 h, depending on how good you want the focusing to be, says Lubman. In the end, you are left with 20 different tubes, each containing liquid-phase protein fractions of varying pI ranges. Once the batch IEF separation is complete, a fraction of the contents from each tube is
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injected into an HPLC system for the second-dimension separation, explains Lubman. In the second dimension, proteins are separated on the basis of their hydrophobicity and molecular weight by reversed-phase HPLC. “But it’s not an ordinary reversedphase HPLC [system],” emphasizes Lubman. “It uses nonporous columns, which make all the difference,” he says. The columns are optimized for separating proteins in the 5to 70-kDa range, which is comparable with most gels and includes many of the proteins currently being analyzed as possible cancer biomarkers. The researchers have even been able to separate proteins as large as 100 kDa, says Lubman. As the proteins come off the column, they are detected by UV and collected for MS identification. To create a protein map analogous to a 2-D PAGE image, the researchers developed original software, which creates a protein pattern from the 2-D separation data. For each fraction, pI is plotted against HPLC retention time. In the image, reversed-phase HPLC peaks are repre-
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sented by different intensity bands, corresponding to the provides much better resolution than 2-D PAGE for prointensity of the peaks eluting off the column. “Because teins
