Two-dimensional gel electrophoresis and the beginning of proteomics.

Featured article: O’Farrell PH. High resolution twodimensional electrophoresis of proteins. J Biol Chem 1975;250:4007–21. No one had heard of proteomics in 1971. The invention of the word was 25 years away (1 ). At the time, doing molecular biology meant studying bacteria or their viruses, the bacteriophage. But as a starting graduate student in Boulder, Colorado, I wanted to study eukaryotic embryonic development. I joined the laboratory of Jacques Pène, which was focused on bacteriophage replication, because he was interested in starting a new system for study. He suggested the colonial algae, Volvox, which undergoes a simple embryogenesis to make two types of cells and a beautifully structured colony. Based on the bacteriophage paradigm, I chose to isolate mutants altered at different points in development. I assessed how the mutants interrupted or derailed morphogenesis, and then I wanted to compare this to how they influenced the changing pattern of gene expression, which, at the time, was best defined by changes in the proteins expressed. The most advanced tool to resolve proteins was the newly introduced Laemmli SDS gel electrophoresis method. However, Volvox and other eukaryotic systems had 100 times as many genes as a bacteriophage. I needed a separation system with comparably higher resolution. Undeterred, I set out to combine the 2 most powerful electrophoresis methods available into a 2-dimensional method. Isoelectric focusing separated proteins by their isoelectric point, while SDS gels separated proteins according to molecular weight. The expected independence of these 2 parameters suggested that if each system could resolve close to 100 proteins, the 2dimensional system should approach 10 000, and that was what I strove to achieve. I also wanted to use highly solubilizing and denaturing conditions so that the method could be applied to diverse biological samples. There were problems galore; nonetheless, even an early effort was sufficiently promising that, based on a single picture and a brief proposal, the National Science Foundation awarded us a small grant to develop the technique. About that time, my advisor, Jacques Pène, left Boulder. I stayed and switched to the laboratory of David Hirsh, where I refined the technique and completed my degree. I got nearly what I wanted. I could not detect 10 000 proteins, but I did see well over a thousand, a level that was pretty spectacular for the time. I used E. coli to test the capabilities of the gels because this system provided tools to characterize performance. I also examined proteins expressed by rabbit embryos with my friend Jon Van Blerkom, and by Candida elegans, the model studied in the Hirsh laboratory. The generality and resolution of the method began to attract a lot of attention. As I started to prepare my thesis, David Hirsh and a fellow faculty member, Larry Gold, came up with the idea of organizing a course to introduce the method to people across the country. I found myself, as a graduate student, teaching a course to some of the prominent scientists of that time, such as Fotis Kafatos and Bruce Ames. But friends and colleagues in Boulder felt excluded, so I did it again for local people. It was a busy summer. I defended my thesis immediately after the courses, submitted my paper to the Journal of Biological Chemistry, and left for a postdoc with Gordon Tomkins in the Department of Biochemistry, University of California, San Francisco. I had been in San Francisco for some time when I got the disappointing news that my 2-dimensional gel paper had been rejected. Apparently, the reviewers did not think it was useful. With the help of some of my “students” who were on the editorial board, a powerful argument was made that the journal should reconsider the decision. Fortunately, they did. The method was put to widespread use. It was the mainstay of my own work for another 4 years before I turned to other research directions (2–5 ). But the big factor that attracted frequent citations was that the paper introduced a new perspective on doing science. There were no “omics” of any sort at the time, not even genomics. It was the first method that allowed a broad molecular perspective, and it provided the early foundation for proteomics. But it took time 1 Department of Biochemistry and Biophysics, University of California, San Francisco (UCSF), San Francisco, CA. * Address correspondence to this author at: Department of Biochemistry, UCSF, Mission Bay Campus, Genentech Hall, S372C, 600-16th St., San Francisco, CA 94158. Received January 9, 2014; accepted January 29, 2014. Previously published online at DOI: 10.1373/clinchem.2014.221630 © 2014 American Association for Clinical Chemistry 2 This article has been cited more than 18,400 times since publication. Clinical Chemistry 60:7 1012–1013 (2014) Citation Classic