Joshua Coon: Retooling Chemical Biology

Profiles provide insights into the lives,backgrounds, career paths, and futuresof scientists who serve as Experts onACS Chemical Biology’s online Ask theExpert feature. Coon will begin answer-ing your questions in mid-May, 2008.Readers are encouraged to submittheir questions to the Experts atwww.acschemicalbiology.org. Theeditors will post the most interestingexchanges on the web site.

T o make groundbreaking discoveriesin any scientific field, researchersneed more than just keen observa-

tional skills; they need a suite of powerfultools. Among the standard tools kept inmany laboratories is the mass spectrom-eter (MS), a machine that is been cast invarious forms since the early 1900s to suitresearchers’ changing needs. Joshua Coon,assistant professor of chemistry and bio-molecular chemistry at the University ofWisconsin�Madison, has made the vener-able MS the focus of his burgeoning career.Coon’s laboratory centers its efforts on solv-ing problems in proteomics, juggling bothbasic and applied projects. He and his col-leagues have reinvented tandem MS into anincarnation especially useful for breakingproteins into manageable peptide segmentswhile preserving post-translational modifi-cations, which could provide useful func-tional clues. Along with collaborators at theUniversity of Wisconsin, an institution re-nowned for its work in stem cell biology,Coon is using this new tool to identify andquantify the array of proteomic changes thattake place in stem cells as they progressalong the path to differentiation. By retool-ing chemical biology, Coon’s work is open-ing the door to any number of exciting futurediscoveries.

From Hobby to Career. Coon was born in1976 in Michigan, the only child of his fa-ther, a high school woodshop teacher, andhis mother, a secretary. Rather than steertheir child toward a particular hobby or ca-reer path, Coon’s parents encouraged hismany interests, especially his interest inbuilding. As a child, he enjoyed construct-ing model airplanes, later taking his hobbyinto bigger structures, including woodenfishing boats.

He remembers having a competitivestreak from an early age. “I didn’t know

what I wanted to be other than good atwhat I did,” says Coon. He recalls going toa fishing shop with his father and talkingwith the proprietor about a special type ofboat necessary to fly fish on a nearby river.Coon and his father chatted about buildingthe distinctive boat, but the proprietor toldthem that it would be impossible for ama-teurs like them to construct. Taking it as achallenge, Coon and his father built three ofthe boats back to back. “If someone saidthat you can’t do something, that it’s toohard, my father helped me think that youcan do anything you want to do,” he says.

A scheduling mishap introduced him tohigher-level chemistry in high school. Be-fore the advent of advanced-placementclasses, Coon received special permissionfrom his school to take math at the local col-lege about 20 miles away. However, whenhe went to register, the math class he wasqualified to take was full. However, a chem-istry class still had some openings. Coon be-gan attending the class, and after a year,he recalls, he was “hooked”. When the timecame to choose a college, he chose an-other local school�Central Michigan Univer-sity in Mount Pleasant, about 30 miles fromhis home�and selected chemistry as hismajor.

Because the school was so close by,Coon lived at home and commuted a half-hour to college, staying on campus to readand study between classes. “Since I was anatypical student, I learned to treat collegelike a job,” he says. Every day, Coon recalls,he showed up at school around 7:00 a.m.and left at about 6 p.m. “I keep the sameschedule now�it’s the same schedule thatI’ve had since I was a freshman in under-grad,” says Coon.

Midway through college, Coon got a cam-pus job running the chemistry department’sMS. Students and faculty dropped off

Published online April 18, 2008

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© 2008 American Chemical Society

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samples of molecules they synthesized,and he made measurements and sent themresults. Coon’s job was purely analytical,he recalls�he did not alter the machine�

yet, he became more and more interested inhow he might improve MS and developnew MS technology. “It played into the sortsof things I liked to do for a hobby,” he says.“The idea of building a new machine tappedinto the idea of working with my hands. Iwas really excited about the prospect of do-ing this for a career.”

Coon had also become interested inteaching after admiring the work of his Cen-tral Michigan professors. To go into teachingand research at a competitive school, heknew that he needed to select a graduateschool carefully. By the time he completedhis undergraduate degree in 1998, Coonhad been accepted into a doctoral programat the University of Florida in Gainesville. Be-sides having a top-rated analytical chemis-try program, Coon says, the school had theadded advantage of being in a warm place.“I was tired of the cold weather, which madeFlorida even more attractive,” he quips.

Big Gamble Pays Off. Early in graduateschool, Coon says he became intriguedwith the idea of working on uncovering thevast amount of information locked in the hu-man proteome, the natural extension of thehuman genome project. “MS was the devicethat seemed best for doing the sequencing.This field was wide open for technology de-velopment,” Coon recalls.

He eventually began working with Wil-liam Harrison, an analytical chemist whodid elemental MS, on a project with a bio-logical bent. Coon’s work focused on takinga sample, such as a peptide or protein, thenusing a laser to put the sample in the gasphase in front of the machine’s inlet. An ionsource nearby the sample generated ionsthat reacted with the substance of interest,giving it a charge. Coon then used the MS tomeasure the sample’s characteristics.

Working with Harrison, Coon says, hepublished four papers on this new tech-

nique within a year and a half (1−4). Thoughit has never been widely adopted, the workserved as an important proof of principlethat such a technique was feasible for mea-suring biological samples.

Harrison encouraged Coon’s interest inan academic career, urging him to aim forthe most ambitious appointment he couldachieve. “His advice was to start as high asyou could get�it’s always easier to go downthan up,” recalls Coon. Knowing that he’dneed a unique postdoctoral fellowship tostay competitive, Coon searched for labora-tories developing innovative ways to use MSfor biological applications. He found the per-fect fit with Donald Hunt, a leader in bio-logical MS at the University of Virginia inCharlottesville.

At the time, Hunt was working with an un-conventional graduate student, John Syka,an engineer who had decided to get his doc-torate after spending decades in a career atThermo Fisher Scientific. Prior to Coon’s ar-rival, Hunt and Syka had already developedan idea for sequencing peptides by reactingpeptide cations with anions in an MS, caus-ing them to fall apart in a way that pre-served the proteins’ post-translational modi-fications. Other more typical techniques forbreaking up peptides for MS analysisstripped these modifications from proteins,removing potentially useful informationabout how proteins are regulated inside acell.

He points out that the idea of reacting an-ions with cations on peptides was not novel;researchers had been trying to do similar ex-periments for the previous decade, butthey hadn’t been successful. Rather thansuccessfully fragmenting peptides, theadded anions instead abstracted hydrogenfrom the cations, leaving the peptides intact.“The literature said that the reaction wewanted to happen wouldn’t work,” Coon re-calls (5). However, he and his colleagueswere not deterred by the challenge. “Wethought that if we chose the anion appropri-

ately and treated it gently, we might get itto work. It was a big gamble.”

Coon and Syka spent the next 10 monthsmodifying an existing MS machine to trytheir experiment, then testing variousclasses of small-molecule anions for thebest candidate for their experiment. Coonrecalls that he and Syka tested the modi-fied machine and their best candidate mol-ecule, a polyaromatic hydrocarbon, on aSaturday. Their first impression was thatthe experiment did not work. “We weredown as low as you can get,” Coon says.However, after averaging the series of scansthe following Monday morning, the re-searchers found hints of evidence that theirexperiment was successful after all.

Coon and his colleagues spent the nextseveral months optimizing the chemical re-action and characterizing their new tech-nique, which they named electron transferdissociation (ETD). Two months after pub-lishing his first papers on this topic (6−8),Coon began applying for jobs. Withinmonths, he was offered a position at theUniversity of Wisconsin�Madison.

Tooling Up as a Team. Coon says thatschool offered him an ideal combination ofcharacteristics to match his goals: running alaboratory that would have a biology focusbut still doing instrumentation and methodsdevelopment. He notes that Wisconsin�

Madison was unusual for having both top-rated biology and analytical chemistry pro-grams. In additional, Coon says, he was im-pressed by the extremely collegial andcollaborative atmosphere at the university,which he’d need to rely on to further his bio-logical work. “All my knowledge of biologyis self-taught. I can talk the talk a little bit,but to do good science, I rely on good col-laborations,” he says.

These attributes sealed his decision; heaccepted a position in the school’s depart-ment of chemistry and began setting up hislaboratory in the fall of 2005.

The past two-and-a-half-years have been“a lot of fun,” Coon says. During that time,

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he has recruited eight graduate students, apostdoctoral fellow, and a laboratory man-ager, who also happens to be his wife. Thetime has also been a productive one forCoon and his new colleagues. “Things havemoved along pretty fast, and I think it’s be-cause of the high-quality graduate studentshere at Wisconsin,” he says. “Everythingwe’ve published has really been on theshoulders of the first- and second-year stu-dents.”

On the basic side, Coon is maintaininghis long-term objective of creating cutting-edge instruments to eventually sequencethe entire human proteome. Last summer,he and his colleagues published a paper de-tailing an extension to the ETD technologyhe’d worked on with Hunt and Syka (9). Thenew work involved modifying an OrbitrapMS machine to enable ETD chemistry, giv-ing measurements with better accuracy andresolving power. He and his co-workers li-censed the new technology to ThermoFisher, which plans to sell Orbitrap ma-chines modified for ETD this summer.

In more recent work, the researchers havefurther optimized the Orbitrap, giving it aphysical source of ions on the back of themachine. They plan to publish on this newtechnology soon.

In his laboratory’s applied projects, Coonis particularly proud of his collaboration onstem cell research with Wisconsin scientistJames Thomson, a pioneer in isolating andculturing both nonhuman primate and hu-man embryonic stem cells. Early in Coon’scareer at Wisconsin, Thomson approachedhim about using ETD to help decipher thefactors that steer these undifferentiated“blank slate” cells into blood, skin, liver,and other committed cell lineages.

The pair and their colleagues have sincepublished a paper detailing the use ofCoon’s advanced MS technology to identifyand quantify various post-translationalmodifications to histones, the chemicalspools that cell nuclei engage to packagelong strands of DNA (10).

The tails of histones can have severalpossible modifications, which serve to in-fluence which genes on nearby DNA areactivated and which are silenced. Previ-ously, researchers have probed for thesemodifications with antibodies, which canreadily locate individual modifications onhistone tails. However, notes Coon, it isdifficult to employ antibodies to identifycombinations of modifications, an impor-tant clue for discovering how these modifi-cations change gene activity. “Each mod-ification is like a word in a sentence,”explains Coon. “If you know all of themodifications, you can read the sentence.But if you just know individual words,you’re missing the context.”

In their paper, Coon, Thomson, and theircolleagues identify 74 patterns of modifica-tions present in stem cells. Strikingly, Coonnotes, proteomic analysis showed thatthese modification patterns shift in charac-teristic ways as cells move from an undiffer-entiated state toward committed lineages.He and Thomson plan to explore and extendthis finding in future research.

Though these two areas of research con-sume most of Coon’s time at the moment,he hopes eventually to tackle a host of otherbiochemical questions. For example, MScan currently detect typical proteins inminute amounts, but the technology oftencannot detect proteins that are consideredaberrant, for example, those that are the re-sults of alternative splicing or SNPs. Coonplans to develop new technology aimedspecifically at detecting these aberrant pro-teins as well as developing new bioinformat-ics tools to identify and characterize what re-searchers will find.

Though he’s expecting plenty of chal-lenges in his future research, Coon predictsthat he and his laboratory will have lots offun along the way. “There’s not a single partabout this job that I don’t like,” he says.

—Christen Brownlee, Science Writer

REFERENCES1. Coon, J. J., Steele, H. A., Laipis, P. J., and Harrison,

W. W. (2003) Direct atmospheric pressure couplingof polyacrylamide gel electrophoresis to massspectrometry for rapid protein sequence analysis,J. Proteome Res. 2, 610–617.

2. Coon, J. J., McHale, K. J., and Harrison, W. W. (2002)Atmospheric pressure laser desorption/chemicalionization mass spectrometry: a new ionizationmethod based on existing themes, Rapid Commun.Mass Spectrom. 16, 681–685.

3. Coon, J. J., Steele, H. A., Laipis, P. J., and Harrison,W. W. (2002) Laser desorption-atmospheric pres-sure chemical ionization: a novel ion source for thedirect coupling of polyacrylamide gel electro-phoresis to mass spectrometry, J. Mass Spectrom.37, 1163–1167.

4. Coon, J. J., and Harrison, W. W. (2002) Laserdesorption-atmospheric pressure chemical ioniza-tion mass spectrometry for the analysis of peptidesfrom aqueous solutions, Anal. Chem. 74, 5600–5605.

5. McLuckey, S. A., and Stephenson, J. L. (1998) Ion/ion chemistry of high-mass multiply charged ions,Mass Spectrom. Rev. 17, 369–407.

6. Coon, J. J., Syka, J. E. P., Schwartz, J. C., Shabano-witz, J., and Hunt, D. F. (2004) Anion dependence inthe partitioning between proton and electron trans-fer in ion/ion reactions, Int. J. Mass Spectrom. 236,33–42.

7. Syka, J. E. P., Coon, J. J., Schroeder, M. J., Shabano-witz, J., and Hunt, D. F. (2004) Peptide and proteinsequence analysis by electron transfer dissociationmass spectrometry, Proc. Natl. Acad. Sci. U.S.A.101, 9528–9533.

8. Coon, J. J., Shabanowitz, J., Hunt, D. F., and Syka,J. E. P. (2005) Electron transfer dissociation of pep-tide anions, J. Am. Soc. Mass Spectrom. 16, 880–882.

9. McAlister, G. C., Phanstiel, D., Good, D. M., Berg-gren, W. T., and Coon, J. J. (2007) Implementation ofelectron-transfer dissociation on a hybrid linear iontrap-Orbitrap mass spectrometer, Anal. Chem. 79,3525–3534.

10. Phanstiel, D., Brumbaugh, J., Berggren, W. T., Co-nard, K., Feng, X., Levenstein, M. E., McAlister, G. C.,Thomson, J. A., and Coon, J. J. (2008) Mass spec-trometry identifies and quantifies 74 unique his-tone H4 isoforms in differentiating human embry-onic stem cells, Proc. Natl. Acad. Sci. U.S.A. 105,4093–4098.

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