AT A GLANCE
Name: Keith Rose
Position: Chief Scientific Officer, GeneProt
Prior Experience: Professor of Medical Biochemistry, University of Geneva, co-founder of Abiotic, which morphed into Ciphergen Biosystems and Gryphon Sciences
This is the first in a two-part interview with ProteoMonitor. Here, Keith Rose talks about his start in protein biochemistry and pre-GeneProt career. Next week he will answer questions about GeneProt and its future.
How did you first get involved in protein biochemistry?
I entered Oxford University as a first year student in ‘71, and in ‘74, after I did three years of courses [in chemistry], there was a project that took a bit less than a year, and for my project I chose protein and peptide mass spectrometry. We went ‘round to see various research workers, postdocs and professors, and that was an exciting thing to do, because it was chemistry applied to biology, which is much more complicated and for me more interesting. Biology for a chemist is interesting. When I synthesize a white powder, I want it to do something; not just make pretty crystals.
How did you analyze proteins by mass spectrometry in those days?
In those days, it was incredibly difficult, and one needed between 0.1 and 1 micromole of a sample to get data. That’s nearly nine orders of magnitude more than you need today! You had to digest anything bigger than about five residues, and you had to do chemical derivitization in order to make the sample volatile — you had to kill all the charged groups.
In those days there were two techniques [to do this]: one was called permethylation, which was practiced best by Howard Morris. The other technique was the reduction of the amide bonds, the peptide bonds, to make what is called polyamino alcohols, and that was practiced by Claus Biemann, one of the most famous mass spectrometrists, at least as far as biochemistry is concerned. Once you had done that, you then either distilled this derivatized mixture of a probe, a direct insertion probe, or you performed a separation by gas chromatography. In those days we didn’t have a computer at the start, so you would watch the chromatogram — you would watch an oscilloscope — to see if it looked as if something was coming out. You’d press a button, and a meter of UV recorder paper would shoot out of the mass spectrometer, and you would have to count all the little peaks manually, and there could be several hundred of them.
Of course it always fell to the student to do that donkey work, so I must have counted every millimeter of hundreds and hundreds and hundreds of meters of recorder paper! The mass scale you recognized through the bleeding of the stationary phase on the GC column, which helped you to make sure you were staying in register as you counted up the mass scale. That was life in those days. Then we got a computer. That helped things a bit.
What kinds of mass specs did you use in those days?
I had a prototype, a VG, as they were called, vacuum generators, [and they] only made about two of these machines. It was called the VG 305. It was a single focusing magnetic sector mass spectrometer, which had a resolution of 1500. It just had electron impact ionization and chemical ionization, because nobody had invented electrospray or FAB, or MALDI, or if they had it was in physics departments and they weren’t being used for peptides and proteins.
What were the factors that led to your going to Geneva?
The factors were that at that time [in the late ‘70s and early ‘80s], it was a difficult period to find funding and a job in England. There was a lot of what they called “dead wood” around, and the “new blood” appointments hadn’t started, so it would have been difficult to find good funding and a good job in England at that time. My boss, Robin Offord, was offered a job as head of the department of medical biochemistry at the University of Geneva, and he wanted to take his best people with him. So I had the opportunity of moving to Geneva, in a new lab, where I was able to hire people and buy machines and so on. For someone who had only done less than two years of a postdoc, it would have been difficult to refuse. Also the fact that the teaching would be in French was a challenge, but also I was able to offer to my wife a French-speaking environment, even though she’s totally bilingual herself. So those were the factors: moving away from chemistry towards medicine, a lot of resources, and I was able to buy a new mass spectrometer, which was called the MS 50 — a huge thing, a double focusing mass spectrometer.
How did your research evolve at the University of Geneva?
I moved to Geneva, and carried on in the area of peptide and protein mass spectrometry. We got the sample requirement down very quickly to 2 to 10 nanomoles, through careful optimization of the chemistry and some improvements on the mass spectrometric side. I was then also interested in something called reverse proteolysis, which was using enzymes to synthesize peptides and proteins, rather than digest them. I found a way of converting porcine insulin into human insulin, which was something a lot of people were trying to do, but what I didn’t know was that Novo in Denmark had put in a patent application. It was one of these submarine patents, where you find out later, so we were never able to exploit what we did, although it worked very well.
At that time, most chemical modification of proteins was done in a non-specific way. One would use a reagent that attacked lysine side chains for example, or iodinated tyrosines or whatever, but it didn’t do a precise modification at a particular place. Now using the chemistry that we had used for converting porcine to human insulin, that is, reverse proteolysis, it was possible to perform site-specific modifications of proteins. It was quite complicated and difficult, but it could be done. We published quite a few papers, and we were involved in several collaborations with companies.
Were there other elements to your research at the time?
That went on up to about 1993, when myself, Robin Offord, Stephen Kent, and a few others, founded what was called Abiotic, which split quite quickly into Abiotic Systems and Abiotic Pharmaceutical Technologies. Abiotic Systems brought in some new technology and has become Ciphergen Biosystems. Abiotic Pharmaceutical Technologies changed its name a few years later and became Gryphon Sciences of South San Francisco.
What we’ve done by putting all that together is we’ve missed one or two little things: one is polyoximes. Part of the chemical modification side of my work was the discovery of the power of oxime chemistry in protein chemistry. Through the oxidation of N-terminal serine and threonine residues one gets a glyoxalyl group which is very reactive towards amino-oxy compounds. These form oximes under extremely mild conditions. In ‘97 we ran a clinical trial, again here in Geneva, with 10 volunteers, of our 22,000 MW artificial malaria vaccine synthesized in my lab. The results were published last year in the Journal of Immunology.