SCIENCE & TECHNOLOGY technical program seems to be stronger than ever. The number of technical papers and poster presentations has been climb ing for many years, and this year a record 2,547 presentations were listed in the pro gram. That's 11% higher than last year's record. In addition to the more tradition al analytical topics, technical sessions ad dressed combinatorial chemistry genomics and proteomics, nanotechnology, and de tection of terrorist weapons.
PITTCON
2002
THE TIMES, THEY AREA-CHANGINV Pittsburgh Conference navigates choppy waters as the instrument industry confronts change RON DAGANI, C&EN WASHINGTON
D
OES PITTCON HAVE THE BLAHS?
Or is it the blues? Or maybe it's just a change of life? It's hard to know for sure. But consider this: Pittcon 2002—the 53rd annual Pittsburgh Con ference & Exposition on Analytical Chem istry & Applied Spectroscopy—lumbered into New Orleans three weeks ago and at tracted only 23,319 attendees. That's down 6.6Φ0 from last year and a whopping 31.6% below the peak-attendance year of 1996, when 34,079 people crowded into the con ference in Chicago. Attendance figures have fallen almost every year since 1996, and they haven't been this low since 1985. Hidden within this year's attendance fig ure is another troubling trend: Slighdy more than half of the attendees were exhibitors. The number of exhibiting companies— 1,144—was down 7.7% from last year and down 10.6% from 2000, when a record 1,280 companies exhibited their wares. On the exhibition floor, Pittcon 2002 seemed to some observers to be more sub dued and blander than in years past. Few er attention-getting ploys were visible, and many of these were retreads from previ ous years. HTTP://PUBS.ACS.ORG/CEN
Nevertheless, Rita M. Windisch, pres ident of this year's show, was extremely up beat about the exposition. She pointed out that the number of conferees (nonexhibitors) actually was up from last year. Af ter the Sept. 11,2001, attacks, attendance at many trade shows plummeted by 20 to 50%, she told C&EN. That Pittcon came away with only a 7% decline is "remark able," she said. "I was relieved and pleas antly surprised at how well we did." Even so, Windisch admitted that "every body's concerned" about the show's slump ing attendance over the longer term: "It's a sign of our economy" Indeed, a recent trade show survey of 40 companies revealed that one-third had cut their 2002 exhibition budgets by up to 35%. Only two respondents said they planned to increase their budgets. There is a bright side, however. At this year's Pittcon, exhibitors told C&EN that, although the number of sales leads was down, the leads were of higher quality— that is, these were potential customers who were more focused, more interested in learning about a particular product, and hence more likely to buy On another positive note, the Pittcon
AS ALWAYSp exhibitors at Pittcon un veiled novel technologies; introduced new or enhanced products; celebrated compa ny anniversaries; and announced recent partnerships, mergers, and acquisitions— and even new advertising slogans. Behind the scenes, instrument companies this year waged an unprecedented publicity blitz us ing e-mail, trying to entice reporters and editors to come to their booths and to nu merous, sometimes overlapping press events. Whether Pittcon is out of sorts or not, it is definitely changing, along with the greater community ofanalytical instrument vendors and users. This changing landscape was the focus of three talks given at the Pittsburgh Conference Breakfast, hosted by Centcom, the company that manages advertising in C&EN and other American Chemical Society publications. This being the 25th anniversary of the perennial event, Centcom PresidentJames A. Byrne put to gether a special program featuring one vet eran Pittcon breakfast speaker and two ed itors whose publications have long covered Pittcon. The veteran was K.C. Warawa, presi dent of K.C. Associates, a market research firm specializing in high technology, par ticularly analytical instrumentation. In her third appearance behind the podium at a Centcom breakfast, Warawa surveyed the changing marketplace. Using data from various sources and studies, she explained how customers, information sources, and services are changing. Warawa noted, for example, that in the pharmaceutical industry, fewer new thera peutic drugs are entering the market, and $35 billion in sales—more than 10% of the prescription drug market—is threatened by expiring patents. In 2000, generic drugs already accounted for 47% of prescription units, and that figure is expected to rise. Because of this, she said, the pharmaceuti cal industry needs to find faster ways to bring drugs to market. High-throughput methods and new technologies should help. In recent years, she noted, there has been "a dramatic change" in the number of C & E N / A P R I L 8, 2002
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SCIENCE & TECHNOLOGY T H E
E N V E L O P E ,
PLEASE
And The Winners Of The 2002 Pittcon Editors' Awards Are...
H
ollywood studios sometimes go to great lengths and expense to promote their Oscar contenders, hoping to sway the people whose votes decide which films get the coveted Academy Awards. A touch of that kind of self-promotional mania surfaced this year at the Pittsburgh Conference with regard to the Pittcon Editors' Awards. These awards—at the gold, silver, and bronze levels—are bestowed each year on the best new products, as judged in an informal poll of journalists and editors covering the conference. With hundreds of new products being exhibited at Pittcon every year, the Editors' Awards represent an effort to develop "a consensus view" of the most newsworthy products, says Gordon Wilkinson, a consultant editor to Instrumenta, a newsletter that covers the global analytical instrument and lab equipment industries. Wilkinson is a prime mover behind the awards. The awards apparently have become prestigious enough that informatics vendor Thermo LabSystems took the unprecedented step of plastering the cover of its Pittcon press kit with a prominent sticker that explicitly asked the recipient to vote for eRecordManager for the Pittcon Editors' Awards. There's no way to know for sure whether that ploy was responsible, but eRecordManager did win the Gold Award, tying for first place with a new product from Jobin Yvon, a division of Japan's Horiba Group. Thermo LabSystems (a Thermo Electron company) touts eRecordManager as a revolutionary new approach for the archiving and management of analytical data. The system, developed jointly with Thermo Galactic, reads and stores more than 150 analytical data file formats that have been used to store all kinds of
people accessing the Internet. In a 1996 study; 2 2 % of respondents had access to the Internet both at home and at work, while 34% had no access. A similar study earlier this year found only 2% had no access at all, while 82% could get on the Internet both at home and at work. Instrument users are finding the Internet "very useful," she noted, for locating information such as troubleshooting tips and part numbers for replacement parts, consumables, and accessories. In the past, Warawa observed, "the reader service card was king."Tbday researchers check print advertising and product catalogs, but they also have a new, more efficient option for finding information: visiting the manufacturer's website. Services are changing as well, and they need to change, Warawa said. Sales begin to fall off during the maturity stage of the product life cycle, so vendors want to extend this stage to increase profits. They can do this, she suggested, by providing customers with more service and support. A n o t h e r breakfast speaker—James F. Ryan, editor of the ACS publications Today's Chemist At Work and Modem Drug 24
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chromatographic and spectral data. It stores the original data file while also automatically converting it into XML (extensible markup language), a platform-neutral format. "Data can be retrieved with no reliance on the original data system, operating system, and hardware," the company points out. The software thus enables organizations that generate and use analytical data to comply with new regulations mandated by the Food & Drug Administration. These regulations, known as 21 CFR Part 11, deal with the requirement for secure archiving and the ability to retrieve records in the future. But beyond that, eRecordManager makes it easier to mine, visualize, share, and compare data from different sources, according to Thermo LabSystems. Data obtained on many older systems, including systems that ran on computers that are no longer being produced, can be given "a new lease on life" by being incorporated into eRecordManager, explains Kevin Smith, the firm's director of electronic record management. The other new product to receive a Gold Award is a combined dispersive Raman and Fourier transform infrared (FTIR) microscope called the LabRam-IR. Developed by Jobin Yvon in collaboration with SensIR Technologies, the LabRam-IR "is the first instrument to offer both Raman and FTIR microscopy in an affordable combination system," according to its developers. The new system consists of a compact FTIR module from SensIR that can be combined with any of the Raman microscopes that Jobin Yvon offers under its LabRam brand name "without compromising the Raman performance," Jobin Yvon says. The Raman and FTIR measurements are made at the same location on the sample, "eliminating the need to transfer the sample from
Discovery—took his listeners on a quick tour of the history of analytical chemistry and how it has changed society He focused on the development of analytical instruments during the past 50 years or so. Dur-
ing this period, Ryan observed, detection limits have steadily been driven lower and lower. In the 1950s, analysts worked at the gram and milligram levels. "In the past 10 or 15 years," he said, "we've gone down to picograms and femtograms and even, in certain kinds of analyses, to attograms {10-18g}." Detection of trace metals has improved from parts-per-million levels to parts-pertrillion and even parts-per-quadrillion levels, he noted. New analytical techniques have had to be developed to achieve these lower detection limits. Environmental concerns and the growth of government regulations have driven many of these advances. Now we find ourselves in the age of genomics and proteomics, Ryan observed. With technologies such as high-performance liquid chromatography, analysts are learning how to measure proteins at the 50-attomole level—a factor of 50,000 times lower than can be routinely achieved with a two-dimensional gel. One of the factoids that one hears, he said, is that all the therapeutic drugs on the market target only 4 0 0 proteins, while HTTP://PUBS.ACS.ORG/CEN
one system to another and then trying to relo cate the measurement point," according to SenslR. The combination of Raman and FTIR spec troscopy in one instru ment allows the ana lyst to access essentially the full range of vibrational transitions, down to 50 cm -1 or less in the Ra man. (The lower limit for FTIR usually is around 600 cm-1.) Garnering the Silver Award was the HR-US BRAINCHILD JEOLs Jun Tamura 101 high-resolution ul trasonic spectrometer shows off the award-winning from Ultrasonic Scien instrument he designed, the tific, based in Dublin, AccuTOF mass spectrometer for Ireland. This instruLC-MS analysis. ~~ ment, which is intend ed for the nondestructive analysis of liquids and complex colloids, uses ultrasonic waves to probe the elasticity and viscosity of sam ples. These characteristics are extremely sensitive to intermolecular interactions, allowing the instrument to analyze a broad range of molecular processes that are difficult or impossible to measure using other techniques, according to the company. The spectrometer can be used to determine the concentra tions of sample components, transition temperatures, enzymatic
each of our cells manufactures 10,000 proteins every day. So this would suggest that there are opportunities for further investigations, "if we could just get down to the point where we could see those pro teins and begin to understand what they do." Ryan concluded his talk by making a very safe prediction that "the analytical in dustry will continue to provide instru ments that routinely and reproducibly gen erate data at low detection limits with automated technologies." PEERING INTO the future is what many Pittcon breakfast speakers traditionally have done, and this year another speaker at the event reminded everyone of that. Relying on back issues of the news magazine she leads, C&EN Editor-inChief Madeleine Jacobs surveyed some of the predictions that have taken flight at the previous Centcom-sponsored break fasts at Pittcon. 'And before you head for the doors," she said preemptively, aware that past breakfast speakers were in the audience, "let me assure you that your prog nostications were amazingly on target." HTTP://PUBS.ACS.ORG/CEN
activities, sizes of particles in suspensions and emulsions, kinet ics of chemical and physical processes in materials, binding stoichiometries and affinities, and other parameters. These types of measurements can be carried out in samples that are optically opaque—a boon to scientists who have been struggling with the problem of analyzing nontransparent samples, says Alan Hulme, director of business development at Ultrasonic Scientific. Ultrasonic analysis is an established technique, but Hulme claims that the new spectrometer can achieve previously unattained levels of resolution (down to 10~5% for ultrasonic velocity). The sample size is typically 1 ml_, but the instrument also can handle samples as small as 30 μΙ_. The Bronze Award went to JEOL's AccuTOF time-of-flight mass spectrometer for liquid chromatography analyses. The company says it is the first mass spectrometer that enables one to accurately measure the mass of analytes present at both low and high concentrations. Typically, analysts have been limited to working within a narrow concentration range to obtain an accu rate mass measurement. The AccuTOF's high, linear dynamic range breaks through that limitation, JEOL says, expanding the applications for mass analysis in the chemical, pharmaceutical, and biotechnology sectors. The new instrument is ideal for measuring the mass of trace components. It offers mass accuracy better than 5 ppm, a resolv ing power greater than 6,000, highly stable tuning and calibration, and single-peak drift correction. Furthermore, the AccuTOF's full-scan sensitivity is an order of magnitude greater than that of magnetic sector or quadrupole mass spectrometers, according to JEOL The four award-winning products were selected from a total of 21 valid nominations—a larger number of nominated products than last year, according to Wilkinson. That suggests that innovation continues to be a driving force in the analytical instrument industry.
Showcasing memorable quotations in memory," the balance of trade may be from industrial, academic, and other no negative, she said. tables, Jacobs pointed out that speakers "The really depressing slide," inJacobs' correctly predicted seven major factors view, was one showing how chemical in that have had the biggest impact on the dustry sales (based on a group of 25 chem instrument business: the business cycle, ical firms that have long been tracked by innovation, growth of the biotechnology C&EN) had declined all through 2001. and pharmaceutical industries, increasing Earnings in each quarter of last year also regulations, automation, the genomics rev were way down compared with the yearolution, and globalization. earlier quarter. The silver lining, though, is that a num In addition,Jacobs presented data from various sources that delineate recent ber of indicators suggest that the overall trends. For example, R&D spending as a recession that started in March 2001 "may percentage of sales in the chemical, very well be over," she noted, adding that biotech, and pharmaceutical industries the economy should pick up in 2002, al over the past decade has been declining, though we may not see any significant she observed. 'And that says a lot about in growth in the chemical industry until 2003. novation in companies." Capital spending Those seven factors impacting the in as a percentage of sales also is declining, as strument business are still in play, Jacobs is industrial employment in chemicals and said. And she harked back to what one allied products. But employment in drug breakfast speaker said in 1980: "These are manufacturing is going up slowly, she not turbulent times—times to make econo ed. "That's really just about the only good mists shake in their boots." That, too, still news you're going to see in these slides." applies, Jacobs suggested. Though it wasn't said for the first time Continuing with the bad news, she showed how the chemical trade balance in at a Pittcon breakfast, one could conclude the U.S. has been falling. "They're pre that the more things change, the more they dicting now that in 2002, for thefirsttime remain the same. • C & E N / A P R I L 8, 2002
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SCIENCE & TECHNOLOGY SMART DUST Small silicon particles such as the ones shown here have been demonstrated for standoff detection of volatile organic compounds.
PITTCON
2002
DETECTING TERRORIST WEAPONS Symposium looks at a variety of sensors for detecting biological and chemical weapons CELIA M. HENRY, C&EN WASHINGTON
W
HEN NEWS OF MAILTAINT-
ed with Bacillus anthracis spores broke last October, no one knew how wide spread the contamination would be. Although the extent turned out to be fairly limited, 22 people—five ofwhom died—were infected with either inhalation or cutaneous anthrax. The events drove home the fact that chemical and biological terrorism are not merely abstract notions. They are real and need to be dealt with. One challenge for people dealing with the threat of terrorism is detecting the agents—sensitively selectively, and rapid ly That topic was addressed at a daylong symposium held at Pittcon last month, where scientists discussed a variety of sen sors being developed for biological and chemical weapons. David R. Walt, a chem istry professor at Tufts University, organ ized the symposium last spring—long be fore the events of Sept. 11,2001, and the subsequent anthrax scare. "This sympo sium is not a case of jumping on the band wagon," he told the audience. Walt told C&EN that even if he had 26
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arranged the symposium in the wake of everything that happened last fall, the speaker lineup would probably have been the same. For each session, he selected a lead-off speaker to establish a context. "It was pretty tough to wean this program to as few people as there are," Walt told C&EN. "I looked for people I knew were going to give good talks and were going to cover some neat technology" David R. Franz—vice president of the Chemical & Biological Defense Division at Southern Research Institute in Freder ick, Md., and former commander of the U.S. Army Medical Research Institute of Infectious Diseases at Fort Derrick, Md. — set the stage for the other talks in the ses sion that focused on sensors for biological weapons. Franz said that in 1996, when he was still in the Army the Department of De fense was "the only game in town" looking at chemical and biological weapons. At that time, Defense was looking for ways to pro tect U.S. troops but didn't really see a need to make a major investment in civilian de fense. That's not true anymore. Billions of
dollars for civilian defense are being re quested for the 2003 budget. The money will be spent on a threat from which five people died, Franz pointed out. "That says something about the unknown nature of the threat," he said. Franz sees biological and chemical weapons as very different beasts. He de scribed chemical weapons as a "hazmat problem" and biological weapons as a "pub lic health problem." An important consideration, Franz said, is whether the goal is to be able to detect agents in order to warn before an attack or to be able to detect in order to treat after an attack. Detecting to warn is difficult be cause of the sensitivity required. Franz recommended that any invest ment be intended to serve dual purposes. "Think public health," he said, "because we may never have another biological event." Discoveries and technologies fund ed as a defense against terrorism can help in the general public health arena. Bernadette Johnson of the Massachu setts Institute of Technology's Lincoln Laboratory described the need for inte grated systems, that is, systems with more than one type of sensor. She said the de tection requirements differ depending on the goal—deterrence, protection, treat ment, or decontamination. IF A SYST Ε Μ is going to provide sufficient information to protect, it must provide a low rate of false negatives and false posi tives, Johnson said. In addition, it must be able to accurately detennine the agent and the source. A number of difficulties com plicate the detection of biological agents, she pointed out. First, the particles are small aerosols. They can have nonspecific signatures, especially if they are encapsu lated. Also, competition from the back ground can swamp the signal from any bi ological weapon. Most detection systems involve staged methods, Johnson said. First a trigger sen sor causes a sample to be collected for an identification assay By that point, the op portunity to detect to warn is pretty much gone. In addition, Johnson said, "no one will trust a single assay," so a confirmatory preferably orthogonal, or independent, as say must be included. The trigger sensor should be broad spec trum, Johnson said, because discrimina tion is not essential at that point. HowevHTTP://PUBS.ACS.ORG/CEN
er, she said, the trigger sensor should not matophores, generally taken from fish, to ly dissecting the various components of trigger an alarm for nonbiological parti- determine whether an active substance is these pathogenic broths, and the cytosencles. It should be flexible so that it can op- present. These cells change their appearance sor is a nearly ideal instrument for scoring erate in different environments and against in response to toxins. This alteration can in- whether one fraction or another has an acdifferent backgrounds. One example of a volve changes in both the shape and color of tive component in it," he said. technology used in a trigger sensor is par- the cells. "Some cells seem to be more senticle fluorescence. Two-channel fluores- sitive to one type of toxin than another," THESE SENSORS detect activity rather cence (ultraviolet and visible) can usually McFadden told C&EN. "One particular than structural information, which distindiscriminate between dirt particles and type of cell can respond in many different guishes them from other sensors, McFadbioagents Johnson said. ways depending on what the toxin is." den told C&EN. "The activity of a subOther speakers described specific techCurrently, McFadden knows that these stance is how it affects a living system. The nologies for bioagent detection. Raymond cells respond to toxins, but he doesn't re- conventional way of determining activity P. Mariella Jr., director of the Center for ally know why "It's clear that a compre- is to expose an animal to a substance and Microtechnology at Lawrence Livermore hensive understanding is down the road, look for symptoms of toxicity," McFadden National Laboratory, talked about im- but a practical use of the cells is here," he said. "Our system is a surrogate for animal munoassays and DNAassays for biological said. "We've been making the assumption testing that is portable and operable by the weapons detection. The Livermore team that we can use the cells by detecting their type of personnel who are familiar with has been building a series of autonomous changes as a practical method for detect- operating instruments." So far, the sensors have been used pripathogen detection systems since 1995, ing active substances. Then, in a few inbased on immunoassays that run on a flow stances, we are investigating the mecha- marily in the lab. McFadden has done some cytometer. The current version runs 10 si- nisms of precisely how the toxins affect testing away from the lab but still in inmultaneous assays based on the Luminex the cells." McFadden told C&EN that his door arenas. "We know the instrument is flow cytometer, including assays for B. an- group is testing sensors made from both a transportable and will operate. There's no thracis, Yersiniapestis (the bacterium that single type of chromatophore and arrays of doubt that the hardware can be transported. The challenge is to transport and causes plague), dndFrancisella tularensis (tu-various chromatophores. store living cells. That's where we've been laremia), as well as simulants, and could be expanded to include even more assays. AN IMPORTANT POINT, McFadden told investing our efforts" to make the instruTo achieve a sensitivity of less than one C&EN, is that his sensor is not intended ment suitable forfielduse. Amore chemical approach to biological particle per liter of air, the Livermore sys- to identify or even quantify compounds. tem uses a large aerosol collector that col- "It can give information on quantity It can detection was described by Wayne Bryden lects several hundred liters ofair per minute. gp/e hints on composition, but its Because typical biological background lev- main purpose is as an alarm," he els are widely variable, the Livermore team said. "My primary goal is to deviews trigger sensors as a stand-alone re- velop a broadly sensitive activity source that could rapidly warn against a detector." massive release rather than as part of an inMcFadden's group has develtegrated system, Mariella told C&EN. oped three generations of senUsing silicon micromacliining, the Liv- sors—dubbed Mercury Gemini, ermore group made a battery-powered, and Apollo—using these chrohandheld thermal cycler for real-time matophores. Mercury is a labDNA analysis using the polymerase chain based instrument that is high BEFORE AND AFTER After being exposed reaction. The performance of the small in- throughput with multiple wells. It to active substances, the chromatophores in the strument is comparable to a conventional resembles an immunoassay plate array (left) change their size (right). instrument, Mariella said. But the detec- reader, he said. The Gemini and tion sensitivity using the micromachined Apollo systems, in contrast, are integrated, of the Applied Physics Laboratory atJohns thermal cycler has reached the lower lim- portable instruments. The cells are kept in Hopkins University He talked about usits of reliability because of the Poisson sam- a self-contained chamber of about a cubic ing miniaturized time-of-flight mass specpling statistics involved. So the Livermore inch. So far, they have not attempted to trometry (MS; see page 34) for bioagent team has developed a microfluidic purifi- miniaturize the electronics. detection. er and concentrator. Ultimately, however, Bryden characterized a strength of MS McFadden finds that the cells respond the size of the autonomous system will be more strongly to the blends of toxins that as also a weakness. "MS can detect anylimited by the aerosol collector, he said. pathogenic organisms produce than to pu- thing, but it can also detect everything. We Philip N. McFadden, associate professor rified toxins. "Our view is that alivingpath- need to separate the anything from the of biochemistry and biophysics at Oregon ogenic organism produces a blend of tox- everything." MS has several advantages as a sensor, State University, described a cell-based ins that in combination exert a powerful biosensor that he calls the SOS (for sto- effect on the cell," he said. McFadden be- Bryden said. MS is rapid—total time to chastic optical signals) cytosensor. This lieves that these sensors can help unravel detection is only five minutes, including sensor uses living cells known as chro- some of the complexities. "We're present- preparation—and sensitive. MS is used to
MS can detect anything, but it can also detect everything. We need to separate the anything from the everything/' HTTP://PUBS.ACS.ORG/CEN
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SCIENCE & TECHNOLOGY look at proteins and other high-mass bioAndrew McGill, head of the functional rate the enzyme organophosphorus hy markers. Bryden showed several examples materials and devices section at the Naval drolase, which catalyzes the hydrolysis of ofwhat MS can do. For example, different Research Laboratory also talked about pat bonds between phosphorus and fluorine, Bacillus species were distinguished based on oxygen, cyanide, or sulfur. The enzymetern recognition. The sensors he described the spectra of spores. Bryden and cowork are based on sorption vapor detection. In catalyzed reactions are specific and sig ers are also working to incorporate tandem these sensors, an analyte-selective coating nificantly faster than chemical hydrolysis. MS, which will provide more detailed is placed on a resonant transducer such as The hydrolysis products can be detected structural information. a surface acoustic wave device. Shifts in the using a number of transduction methods, device resonant frequency that occur when False alarm rates are still a problem to be including electrochemistry and optical target compounds are sorbed to the poly dealt with, Bryden noted. A detector that spectroscopy mers coating the surface allow the detection takes a reading every five minutes and has The enzymatic biosensors can also be κ of vapors and gases. Arrays of dea 1% false alarm rate will trigger 5 vices coated with different sorptive three false alarms per day—an un d polymers are needed to generate acceptable level, Bryden said. | "nose" prints of gases. These patTiming to chemical weapons de Ϊ terns, when combined with pattern tection, Walt said in his introduc % recognition techniques, allow idention that an ideal detector would be | tification of target analytes. able to measure everything, cost S McGill and coworkers improved nothing, be easily manufactured, < the performance of the devices by and be ubiquitous—an ambitious switching from polymers with oxy goal indeed. Cross-reactive sensors gen in the backbone (which can ad are necessary, Walt said, as well as sorb water) to polycarbosilanes, what he called "smarter sensors" with increasing densities of active that would not so much identify a sites for nerve agents. They en substance as answer the question hanced the sensor performance by "Is it bad?" an order of magnitude simply by MichaelJ. Sailor, a chemistry and "tweaking" the backbone and the biochemistry professor at the Uni arrangement of the functional versity of California, San Diego, de groups, McGill said. scribed his group's efforts in mak VISIBLE CHANGE The right side of this porous ing sensors using porous silicon. The silicon wafer has been exposed to HF, which etches A prototype of the chemical sensors are based on color changes the silicon and changes its optical properties. agent detector, called pCAD, has that occur under different chemical ~~~~ already been taken into the field, conditions. One sensor incorporates a cat McGill said. The array responds on the mil used to detect and identify organophosphate alyst that hydrolyzes P-F bonds, a charac lisecond timescale, reaches an equilibrat pesticides. These compounds, which are teristic of the class of nerve agents that in ed response in less than half a second, and readily available commercially are used as cludes sarin and soman. W h e n the P-F returns to baseline quickly as well. The simulants for chemical warfare agents in ex bond is hydrolyzed, hydrofluoric acid is real-time detection limits of the device periments. However, because these com produced, which etches the silicon surface have been demonstrated to be at miosis pounds are so readily available and are also and causes a change in the optical proper concentration levels. Miosis is the first clin neurotoxic, Mulchandani noted that per ties (C&EN, June 12,2000, page 12). ical effect that nerve agents cause, which haps we should be more concerned about results in a constriction of pupils at ex Sailor also described sensors made from them being used as terrorist weapons than tremely low concentrations and could af nanostructured "smart dust." "We take less accessible agents such as sarin or soman. fect pilots. McGill and coworkers hope to these films, and instead of putting them on chips, we break them up into really small TO IMPROVE the specificity of the biosen shrink the pCAD from its current ciga rette box size to a smaller matchbox-size sors, Mulchandani and his coworkers are dust particles. We structure the films so detector, called the Beaglette, which could using directed evolution to mutate the en that they will return very specific wave zymes. The mutants respond differently be used as a disposable device. lengths of light at very specific angles. to various compounds. Arrays of these mu Array sensors are "dumb" without train These films turn colors based on what's in tants can be combined with pattern recog ing, McGill pointed out. The interferences them." Using the particles, a helium-neon nition to distinguish between compounds. expected for a given application are an im laser, and a telescope, Sailor and his col These mutations don't need to be near the portant part of that training. The range of leagues were able to detect ethanol, ace enzyme's active site, Mulchandani said. potential interferences increases greatly tone, and toluene vapor from a distance of 2 0 meters —the first step in using the By combining organophosphorus hy when the application is homeland defense rather than military "The answer," McGill smart dust for detection at a distance, or drolase with microchip separations, one said, "is not a single technology, but or "standoff" detection. can also use the biosensors to identify thogonal technologies." chemical warfare agents and organophos More traditional biosensors, which use phorus pesticides. Mulchandani and his Walt reiterated that point in summing enzymes to detect, are also being devel coworkers are working on lab-on-a-chip up the symposium. Developing detectors oped for chemical agent detection. Ashok technology that will detect both explosives for terrorist weapons will require a multiMulchandani, a professor of chemical and and chemical nerve agents. They have re disciplinary effort, he commented. "There environmental engineering at the Univer ceived a grant from the Department ofJus are vexing problems still facing us, such as sity of California, Riverside, described the tice to develop the technology speed and sensitivity" • development of biosensors that incorpo 28
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SCIENCE & TECHNOLOGY PITTCON
2002
A PROTEOMICS FLOWER BLOOMS AT PITTCON Chemists are embracing the analytical challenges of proteomics research in a big way STU BORMAN, C&EN WASHINGTON
received the first Pittcon Achievement Award, honoring outstanding achievements during the early stages of one's independent scientific career. Clemmer and coworkers are developing gas-phase ion mobility strategies for proteomics use. In ion mobility spectrometry (IMS), mixtures ofions are separated in agas as they traverse an electricfieldin a "drift tube." The separation is based on differences in the ions' charge, mass, and shape. Clemmer's group uses IMS as a highly parallel way to prepare samples for analysis by time-of-flight tandem mass spectrometry (TOF-MS/MS). "Our strategy is to take whole cells, digest the cell proteins to form peptides, separate the peptides by standard liquid chromatography (LC)—yielding typically 10 0 or morefractions—separatethem in a drift tube by IMS, and then analyze them by reflectron time-of-flight MS/MS," Clemmer said. The multidimensional technique is thus called LC-IMS-TOF-MS/MS. It makes it possible to carry out MS/MS experiments in parallel—that is, more quickly—whereas "up to now it's been a serial process," he said.
ROTEOMICS CLEARLY REP the proteins are in cells and how they inresents the next major de- teract with other molecules and each othvelopment stage in under- er. It poses a lot of challenges for analytistanding the mechanisms cal chemists." of life, following the comNovotny, who spoke at a Pittcon 2002 pletion of the Human Genome Project," symposium titled 'Analysis of Complex said chemistry professor D. Jed Harrison Systems," exemplifies the growing numof the University ofAlberta, who arranged ber of analytical chemists who are inaPittcon2002 symposium on proteomics creasingly embracing the analytical chalapplications of chemical microinstrumen- lenges of proteomics. His group recently tation. 'Analytical chemists have recognized joined the Indiana Proteomics Consorhow important the contributions of their tium, a newly established collaboration field were to the sequencing project, and arnong researchers at Eli Lilly Indiana Unimany appear to have reached the conclu- versity, and Purdue University that aims to sion that they could play a significant role develop novel analytical techniques and in the burgeoningfieldofproteomics, too." instruments for proteomics. The complex There's been an evolution toward the use systems symposium was arranged by asso- THIS TYPE of multidimensional separaof proteomics "to look at many proteins at ciate professor of chemistry Adrian C. tion is analogous to lining up the residents of New York City by height, separating once," said professor of chemistry and bio- Michael of the University of Pittsburgh. chemistry Catherine C. Fenselau of the UniThere are 50,000 to 200,000 proteins them further by weight, and then distinversity of Maryland. "This will have enor- in the human proteome, noted chemistry guishing among them once again by hair mous implications for science and will create professor David E. Clemmer of Indiana color, Clemmer explained. "Y>u can sepaa range of new business opportunities." University, and analytical chemists are try- rate alot ofpeople pretty effectively by usFenselau said she recently saw an ad ing to develop better ways to separate and ing just these simple characteristics." LC-IMS-TOF-MS/MS actually pro"stating that big pharma was now going to identify them. At the session, Clemmer examine every protein in cells to z vides four degrees of separation, be§ cause MS/MS is two-dimensional. discover new drugs. How will uniο versities participate in this effort?" S "We think we have the capacity to she asked. For one thing, "there will £ separate 10 million peptides," he be an increase in proteomics cenS predicted. ters with shared facilities" to disI Clemmer and coworkers recent tribute the high costs ofproteomics ly used the technique to compare instrumentation among many users. peptides from stressed and nonAnd Fenselau noted that the Hustressed cells after derivatizing them man Proteome Organisation has with two different isotopic labels been formed to coordinate pro{Anal. Chem., 74, 950 (2002)}. Be teomics efforts internationally yond Genomics, a Boston-based "We can hardly keep the procompany Clemmer cofounded, is teomics students we're training in also planning to use it in proteomics the lab," she said, "because there's studies. such aggressive recruitment for "One of the major complications them. So we need to train more of analyzing proteomes is that a lot students." of proteins are glycosylated," said And according to chemistry proNovotny in his presentation at the fessor Milos V Novotny of Indiana session. Glycosylated proteins par University, Bloomington, "Proticipate in a multitude of biochem teomics will be with us for a long ical interactions and play an im time. Besides structural identificaportant role in immunity and tion of proteins and their quantifi- NEW AND IMPROVED Speicherand coworkers' disease, but researchers "are only cation, it is concerned with where pref ractionation technique improves 2-D gel analyses, at the beginning of understanding
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SCIENCE & TECHNOLOGY the physiological functions of glycopro teins," he said. Having the ability to analyze the struc tures of oligosaccharide groups on glyco sylated proteins would help, but it remains a tremendous challenge. Glycosylated pro teins are enormously complex, each type is typically present in cells in vanishingly small amounts, and no technique like poly merase chain reaction (used to amplify DNA) is available to amplify them. 'Ana lytical chemistry advances will be needed to address this problem," Novotny said. "There's a need for new instrumentation that is faster and more informative." He and his coworkers are developing microscale enzymatic cleavage procedures to remove oligosaccharides from glycosy lated proteins and novel capillary electrochromatography (CEC) media to sep arate the cleaved sugar groups [Chem. Rev., 102,321(2002)}. They recently isolated glycosylated pro teins from the tiny vomeronasal organ of rodents, which senses pheromones. They washed the proteins, separated them on gels, isolated the spots, removed the 0- and N-linked oligosaccharides, separated them by capillary electrochromatography, and thenanalyzed them by MS/MS. "We found major differences between male and female mice in expression levels andglycosylation patterns of vomeronasal proteins," Novot ny said. "These distinctions can potentialWELL EQUIPPED Clemmerwith LC-IMS-TOF-MS/MS instrument.
Kennedy, graduate student William E. Haskins, and coworkers have been devel oping a high-sensitivity capillary LC-ESIMS/MS technique that helps address these problems. The technology is based in part on earlier work by biochemistry professor Richard M. Caprioli and co workers at Vanderbilt Uni versity School of Medicine, who developed miniaturized ESI systems and high-sensi tivity ESI-MS/MS methods for analyzing LC-separated compounds. Kennedy's group has now miniaturized LC columns and ESI devices still PEPTIDE neurotransmitters, further for use in trace neusuch as Met-enkephalin, tend Ν 0 VO t η y ropeptide analysis. to be present at extracellular Kennedy and coworkers have improved concentrations only a hundredth or thou sandth of those of small-organic neuro LC-ESI-MS/MS detection limits from transmitters, such as glutamate, dopamine, about 100 attomoles to the 2- to 5-attoand 7-aminobutyric acid. Neuropeptides mole range and have demonstrated that can be detected in vivo by radioimmuno their system can identify endogenous neu assay (RIA) and enzyme-linked immuno ropeptides at those levels by their MS/MS sorbent assay (ELISA), but the approxi fragmentation patterns [Anal. Chem., 73, mately 100-attomole detection limits of 5005 (2001)}. At Pittcon, Kennedy re these techniques are too high to detect pep ported preliminary results of a collabora tide neurotransmitters without necessitat tive study with assistant professor of psy ing inordinately long sample collection chiatry Robert E. Strecker's group at Har times. RIA and ELISA also require that vard Medical School in which differences you know in advance what peptide you're in the chemical composition of extracel looking for, making them inapplicable to lular regions of rat hypothalamus tissue the analysis of unknown peptides, and were detected in sleeping versus alert states. Chemistry professor Jack Beauchamp they're limited to the determination of one peptide at a time, making them impracti and coworkers at California Institute of Technology are developing biomimetic cal for use in a proteomics environment. reagents to cleave proteins selectively in the gas phase prior to sequencing them by MS. The goal is to be able to characterize proteins in the gas phase using MS with out having to first use a solution-phase process like tryptic digestion to cut them into manageable fragments. The researchers have designed a number ofreagents that bind to specific sites on pro teins and then induce cleavage at or adja cent to those sites. For example, the ligand 18-crown-6 ether forms noncovalent com plexes with proteins by binding to lysine residues with high specificity [Int. J. Mass Spectrom., 210,613 (2001)}, while the larg er 30-crown-10 ether preferentially binds to arginine, even in the presence of lysine. Such ligands can be converted into "lar iat ethers"—crown ethers with attached ly be related to differences in male and fe male responses to pheromones." Chemistry professor Robert Τ Kennedy and coworkers at the University of Florida, Gainesville, are also develop ing novel analytical methods for studying neurological pep tides. They use in vivo sampling techniques, capillary LC, and electrospray ionization (ESI) MS/MS to studvtiny quantities of peptide neurotransmitters in the brain—to identify neu rotransmitters released during stimulation, for example.
"Analytical chemists have recognized that they are well situated to play a key role in developing the tools required to succeed in this complex enterprise." 30
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acidic groups capable of attacking the pro tein at or near the binding site. But the en ergy required to cleave the protein is gen erally greater than the binding energy of the Hgand-protein complex. So when the ligand tries to induce cleavage, it ends up falling off the protein instead of cutting it.
neously cleaved by proteolytic enzymes and isotopically labeled. Proteinsfromdifferent cells can be labeled with different isotopes, making it possible to analyze them togeth er in a single run and determine the con centrations of proteins from different sources. Fenselau's group recently used high-performance LC (HPLC) andMS/MS BUT BEAUCHAMP and coworkers, in col to study changes in protein expression in laboration with Caltech assistant professor different groups of human cancer cells. of chemistry Brian M. Stoltz's group, re Using MS as a readout, there are two cently prepared lariat ethers containing general classes of approach to differential two crown ethers with a cleaving agent in quantitation of protein levels and protein between, and some of these cut proteins modifications for proteomics studies, ex successfully Once the technology matures, plained chemistry professor BrianT. Chait Beauchamp hopes it can be used to facili of Rockefeller University, New York City tate protein sequencing and to achieve se "One uses metabolic labeling with heavy lective recognition of functional protein stable isotopes, while the other uses some motifs. kind of chemical labeling," such as isotopeAnother proteomics symposium, coded affinity tagging (ICAT). "They each "Comparative Proteomics for Studies of have pros and cons." Chait reported on a Cell Function," was arranged by Fenselau. metabolic route to protein labeling in It focused on the determination of differ which cells are grown in an isotopically en ences in protein expression in different cell riched culture medium. types, such as healthy versus sick cells. Assistant professor ofcell biology Steven Postdoc Xudong Yao explained at the Ρ Gygi of Harvard Medical School helped session that Fenselau's group has developed develop the ICAT strategy when he was a a technique in which proteins are simulta postdoc with proteomics researcher Rue-
di Aebersold, who is now at the Institute for Systems Biology in Seattle. ICATis a chem ical isotope labeling technique that is used "to compare on a very large scale the dif ference between protein levels for two dif ferent cell states," Gygi said. He and his coworkers recently devel oped another labeling strategy called AQUA (an acronym for "absolute quanti tation") that measures a protein's absolute, as opposed to relative, abundance. In this technique, a chemically synthesized ver sion of a targeted protein is used as a stan dard to determine the absolute amount of that protein in a biological sample. Gygi and coworkers recently used it to obtain the absolute quantification ofphosphorylated versus nonphosphorylated protein directly from total cell lysates. Chemistry professor Fred E. Régnier and coworkers at Purdue University use a combination of deuterium labeling and affinity selection to identify proteins in cancer cells that change concentration in response to chemotherapy "The big difference between ICAT and our approach is that we can target posttranslational modifications in addition to protein exprès-
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[email protected] SCIENCE & TECHNOLOGY highly specific manner," MacBeath said. "Multiplexed screening gready increases the number of interactions that can be studied, dramatically decreases sample consumption, and enables us to assess the specificity of compounds during the screening process." The group is also using microarrays of antibodies "to study how cell-permeable compounds modulate both the abundance and posttranslational modification state of multiple proteins simultaneously" MacBeath said. "These and otherprotein profiling technologies provide a more holistic understanding of how bioactive compounds modMULTIDIMENSIONALClemmerand ulate complex protein netcoworkers generated these LC-IMS-MS plots of works within the context of a mixture of peptides from digestion of 18 a living cell." proteins. IMS spectrum of peptide ion from aldolase is highlighted in the ion mobility plot. Lubman and coworkers LC-IMS-MS generates some 500 mass spectra combine 2-D liquid separafor each chromatographic slice (red bar) — tions with MS to profile demonstrating the extremely high resolving proteins in different types power that makes it very promising for of tumor cells. They sepaproteomics analysis. rate proteins using isoelectric focusing or chromatosion," Régnier said. "This is critical in many focusing and separate them further by diseases." His group was able to detect "15 reversed-phase HPLC. They then use to 20 specific proteins associated with non- TOF-MS to obtain the molecular weights Hodgkin's lymphoma in the serum of dogs of the separated proteins. The result is a 2and to show that their concentration ei- D "mass map" that's analogous to a 2-D gel ther decreased or increased with but has much higher mass accuracy— typchemotherapy" he said. "This was possible ically in the 100-parts-per-rnillion range. because stable isotope technology is away tofindneedle-in-a-haystack-type proteins." A third proteomics symposium at Pittcon—"Cancer Diagnostics Based on MS, Protein Chips, and Microarrays for Detection of Biomarkers"—also focused on the proteomics analysis of different types of cells, in this case using protein chip technology The symposium was arranged by chemistry professor David M. Lubman of the University of Michigan, Ann Arbor. MICROSPRAYER This combined Gavin MacBeath, who will join Harvard capillary LC column and electrospray University's department of chemistry and emitter tip is a key part of the LC-ESIchemical biology as an assistant professor MS/MS system designed by Kennedy this July, reported at the session on tech- and coworkers for trace neuropeptide niques to discover small organic molecules analysis. capable of modulating protein function.
