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20 Years Protein Experience Gives Hunt Confidence in Proteomics

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AT A GLANCE

Name: Donald F. Hunt

Age: 60

Position: University Professor of Chemistry and Pathology, University of Virginia, and scientific advisor to MDS Proteomics

Prior Experience: First to adapt triple-quadrupole mass spectrometry for sequencing peptides in mixtures; pioneer in the development of nanoflow HPLC, electrospray ionization mass spectrometry, and FTICR mass spectrometry for peptide sequence analysis

Don Hunt may not be looking for publicity, but it seems to have found him. The University of Virginia mass spectrometrist has won no fewer than eight awards over the past 12 years from such organizations as the American Chemical Society and the Protein Society for his contributions to analytical chemistry and protein analysis, and his research seems likely to continue making waves for years to come.

Hunt’s success has much to do with his talent for science, and perhaps also with his stubborn self-confidence. Flunking out of Stanford University as a first-year graduate student in chemistry during the early 1960s didn’t stop him from going on to earn a doctorate in organic chemistry from the University of Massachusetts, and ultimately a faculty position at U. Va. Nor could public scrutiny in front of 500 protein scientists at the 1988 meeting of the Protein Society shake his belief that he had correctly determined the amino acid sequence of a synthetic peptide of novel structure using only mass spectrometry — a feat most scientists at the time believed was only remotely possible.

 

Doing Everything

 

But while Hunt never earned a degree from Stanford — he failed to pass all of the entrance exams for the chemistry department — “it was there that I decided to become a faculty member.” To achieve this aim despite falling off the academic track, he returned to his native Massachusetts to study organometallic chemistry at U. Mass., and went on to post-doc for Klaus Biemann at MIT in mass spectrometry.

While working in Biemann’s lab, one of Hunt’s first challenges was to sequence oligonucleotides by mass spectrometry. “We published one paper where we got up to three nucleotides,” he said with a laugh. Later, after moving south to U. Va. to take a faculty position in 1968, Hunt got caught up in organometallic chemistry research, and supervised the university’s mass spectrometry lab when it became only the third recipient of a magnetic sector mass spectrometer [an instrument called an AEI MS9] outfitted for chemical ionization. After several years of instrument development and forays into environmental chemistry, he thought he might try his hand at sequencing proteins, because “when you’re in your thirties you think you can do everything,” he said.

 

Don’t Know Much About Proteins

 

But Hunt didn’t know anything about proteins at the time, so he and one of his graduate students developed a collaboration with Howard Morris at Imperial College, London. Morris taught them permethylation, a technique for replacing all the hydrogen-bonding structures with methyl groups that made the peptides volatile. For the first several years Hunt’sefforts to sequence proteins met with little success, primarily because the biochemistry department at U. Va. had supplied his lab with a drastically mischaracterized protein. Instead of having a molecular weight of 12 kDa, as the biochemistry department had indicated, the protein, apolipoprotein B, actually had a weight above 500,000 kDa, meaning every time he weighed some out he had 40 times less than he thought he had.

Once he overcame that obstacle, Hunt put his group’s expertise in mass spectrometry instrument development to good use. A graduate student in his lab, Jeffrey Shabanowitz, had built one of the first triple-quadrupole spectrometers in the late 1970s, and he and Hunt demonstrated how to apply the instrument to sequencing complex mixtures of peptides using an early form of tandem mass spectrometry. In the early 1980s, Hunt’s group also figured out how to adapt the fast atom bombardment technique for ionizing peptides to the triple quadrupole and then published a paper that outlined how proteins could be sequenced with this combination of techniques. In 1986 his group finally published a paper describing how they had used this method to generate partial sequence information on the infamous apolipoprotein B. With this approach, sequencing peptides took only a few hours.

Hunt, however, still had to convince the protein chemistry community that triple-quad mass spectrometry was the preferred method for analyzing proteins. Up until the mid-1980s many mass spectrometrists had their hearts set on the more expensive and hulking magnetic sector instruments, and in addition, many protein chemists believed that sequencing proteins via chemical means — by Edman degradation — was superior to any new-fangled triple quad mass spectrometry-based approach. But, following Hunt’s successful defense of the method at the 1988 Protein Society meeting, many in the field came around to his camp. “Up until the mid-80s the biochemical community did not believe that you could sequence proteins or peptides by mass spectrometry, and it wasn’t until 1986 or 1987 that we could show that we could do it both faster and with higher sensitivity than the Edman people,” he said. “That began to turn the tide.”

 

New Mass Spec Methods

 

In the early 1990s Hunt turned his attention to immunology, and developed mass spectrometry methods for analyzing mixtures of class I and II peptides presented to the immune system on the surface of human cells. He also became a founder of a company, Receptor Laboratories, in an effort to develop vaccines against cancer using mass spectrometry to identify T-cell antigens on the surface of cancer cells. Although Receptor ultimately failed as a company, Hunt realized that the technology he had developed for studying peptides on the surface of cancer cells could also find more general application in analyzing proteins. To make this happen, he started a proteomics institute at U. Va. in 1999 and with the support of Novartis and Thermo Finnigan continued to develop new methods and instrumentation, including nanoflow HPLC interfaced to Fourier transform mass spectrometry, for the selective analysis of phosphoproteins in complex mixtures and for studying differential expression of proteins in cells.

Hunt had decided to populate his proteomics institute with former members of his academic lab, but the problem was he couldn’t hold them in the face of more lucrative opportunities elsewhere. His solution was to form a company, but he didn’t have the informatics and automated sample preparation expertise to create a viable full-scale proteomics business. When MDS Proteomics, a spin-off from Canadian health services giant MDS, approached him in 2000 about joining forces, Hunt decided it made sense to sacrifice the independence of a company of his own. “I could have started my own company but I would not have been able to compete with some of the larger established entities,” he said. “[MDS Proteomics] already had a deal with IBM so they had all the computing power and software, and they had this whole protein-protein interaction thing automated. The automation, informatics, and the BIND database were more than I could overcome, so I would have been relegated to a niche.”

 

Proteins as Bait

 

Since early 2001, the Charlottesville arm of MDS Proteomics has grown to a 10-person operation and will soon move into a new facility that will allow additional expansion, Hunt said. Hunt and his group are now applying their technology for analyzing phosphoproteins and differential protein expression to the company’s efforts to find novel cell-surface markers, protein-protein interactions involved in signal transduction pathways, and new, validated drug targets in general.

“If you take a drug or its protein partner and use it as bait, you can then pull out all the proteins that interact with either and understand the entire interaction pathway in the cell,” he said. “The idea is you know everything that’s triggered in this pathway and you can then develop drugs that produce the desired result while minimizing harmful side effects.”

Hunt admits that the lead time for translating proteomics into therapeutics may span 5-15 years, and that many of the companies and academic investigators in the field have inflated expectations, but he’s convinced that the proteomics approach to drug discovery and development will ultimately be a huge success: “The bottom line is that almost all drugs target are proteins, so I cannot imagine that the pharmaceutical field itself is not going to be revolutionized by what we and others are presently doing in protein chemistry.”

— JSM