The pre-omics era: the early days of two-dimensional gels

Abstract

I present a personal view of the beginning of two-dimensional gels and unsanctioned proteomics. I was still a young graduate student in the early 1970s when I developed methods for two-dimensional gel electrophoresis that became widely used. Though the method gave us the capacity to do things that had never been done, the value of global enumeration of proteins was not appreciated, and we were still two decades away from the invention of the term proteomics. I describe a period of exploration where, by exercising our new capability, we conducted the first proteomic type expression experiments, and made unforeseen contributions to advances in biology. Detection of single-amino acid substitutions validated genetic selections in cultured cells, and revealed a regulatory system that maintains the accuracy of protein synthesis by assuring an unbiased supply of its substrates. We documented biologic control with a dynamic range >10(8) fold, and, in a surprising turn, we identified an approach that provided a major breakthrough in recombinant DNA technology, the ability to express cloned sequences in Escherichia coli. The challenge then and now is to use a capability for global analysis to produce new insights into fundamental aspects of biology and to drive substantive technical advances.

Figure 1

Images of the development of Volvox carteri. The asexual egg or gonidia (A) undergoes cleavage (B–D), producing large reproductive cells on the outside of a shell of numerous small flagellated somatic cells (E) and then a morphogenetic process called inversion (F), which I like to think of as a primitive version of gastrulation, inverts the organism to put its reproductive cells on the inside of the colony. This development normally occurs within a mother colony and (G) depicts mature colonies with developing young on the inside. Note that the adult is ball of about 4000 flagellated somatic cells organized so that flagellar beating of all of the cells produces a concerted force that rotates the colony and drives it forward toward a light source. The reproductive cells and their development occur inside this protective shell.

Figure 2

My first two-dimensional gel. Much of the early work in getting the gels working involved development of ways to run good IEF separations in presence of denaturants that would allow me to run whole cell preparation. After fussing with approaches for a few months this is what I obtained in my first attempt at adding the second dimension. The image is an auto-radiogram of E. coli proteins labeled by growth in the presence of ¹⁴C labeled amino acids. The isoelectric focusing dimension is on the horizontal axis with the basic side to the left, and the SDS separation displays high-molecular weight proteins toward the top and low toward the bottom. This is the orientation that I have always used, but others have chosen to change it creating a problem of standardization. The separation is compromised by clustering of the proteins in the center of the IEF dimension, by streaking, and by fuzzy spots. I was later able to improve these aspects.

Figure 3

This gel was control (AMP added) from a large set of gels that I ran May 27, 1974 as part of the analysis of cyclic AMP responses in E. coli. The sample was wild-type E. coli K12 (strain 1100) grown in minimal glucose medium and labeled with ¹⁴C-amino acids. The gel was prepared and run as described [16] using a 10 to 14% exponential acrylamide gradient in the second dimension. A number of experiments carried out at this time gave reproducibly excellent results.