Proteomics: characterizing the cogs in the machinery of life - Focus

Environmental Health Perspectives, Nov 15, 2003 by Ernie Hood

Now that human genome sequence is complete, the quest to extract beneficial knowledge from it is on. One of the most promising, active areas of exploration lies in the human proteome--the global expression of proteins, those marvelouus strings of amino acids responsible for all human biologic processes. Proteins are life, and the recently developed ability to study them on a large scale, quantitatively and qualitatively, is known as proteomics.

The proteome may never be completely solved in the same way the genome was. The genome is relatively static, and presented a finite end point. The proteome is dynamic, changing constantly with time and conditions, with proteins interacting to networks and pathways to respond to stimuli and carry on the endless business of cellular function. The challenge of completely mapping the proteome is widely considered to be several orders of magnitude greater than that of the genome. The picture is so complex and so dynamic that some proteomics experts question the very concept of the existence of a measurable human proteome. Famed genomicist J. Craig Venter put this doubt succinctly when he told the 5 April 2001 Wall Street Journal that "there ain't nosuch thing as a proteome."

Nevertheless, there can be no debate that proteomics is poised to deliver vast amounts of useful information about physiologic function at the subcellular, cellular, organic, and systemic levels, yielding profound new insights into disease and drug mechanisms, the effects of environmental exposures, and much more. Although a comprehensive map of the entire human proteome may never be accomplished, protein maps of human organs, glands, and fluids and of entire less-complex organisms are within sight, and major efforts are under way to document many of those proteomes.

Evolution of Proteomics

In large measure, proteomics has emerged in parallel fashion with the other "-omics" fields such as transcriptomics and metabonomics. The technologies, methodologies, and grand ambitions of the Human Genome Project have rapidly proliferated and now permeate virtually every area of the life sciences.

Just as the advent of genomics brought the ability to discover large numbers of genes quickly, proteomics was born when technologic advances allowed scientists to widen their focus from the painstaking isolation and identification of single proteins to a more comprehensive view of the entire protein complement expressed in a given cell line, tissue, or organism. However, proteomics researchers employ their own unique mix of tools, approaches, and skills to address the questions they seek to answer.

Although the term "proteomics" did not exist until 1994, when Australian postdoctoral student Marc Wilkins coined it, the practice of the science has been going on since the mid-1970s. Two milestone technologic breakthroughs facilitated the ability to look at multiplicities of proteins, both of which, although much refined, are still in wide use today in laboratories around the world.

At its core, proteomics is all about separation and identification--the process of taking a sample of interest, separating out all of the proteins therein, and then identifying them. The first major breakthrough, which was a great leap forward in separation, took place in 1975 with the introduction of two-dimensional gel electrophoresis (2DE).

With this method--still the first step in many proteomics experiments--proteins from a sample are separated on a polyacrylamide gel according to their mass and charge, which, along with intensity, are what provide the spectrum that makes up a protein's distinctive signature. The more abundant the protein, the larger and more intensely staining the spot on the gel.

The only problem is that 2DE, while it allows separation and visualization of the protein complement, does little or nothing to address identification. Regardless, the advent of 2DE was so exciting that in 1980 it spawned the proposal of a Human Protein Index project--an effort to catalog all human proteins and then use that knowledge to define the genome (although Congress considered the project, it was never funded, and advances in genomics soon bypassed the idea).

The second major breakthrough, which really brought proteomics into its own, was the arrival of two crucial techniques in the 1980s that made possible the use of mass spectrometry (MS) to identify proteins: matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). These methods allow protein samples to be ionized for analysis in a mass spectrometer, producing a pattern called a mass spectrum. These mass spectra--which often number in the thousands for a given sample--can then be used to positively identify proteins or protein digests (strings of peptides or protein fragments produced when the proteins are ionized) through the automatic querying of protein databases. Unidentified or novel proteins can be analyzed through further MS runs or by other techniques.

"ESI and MALIDI were a quantum leap," says William Pierce, a professor of pharmacology, toxicology, and chemistry at the University of Louisville School of Medicine. The subsequent development of time-of-flight (TOF) detection, which expanded the range of ionic molecular weights detectable by MS instruments, brought further analytic capabilities to MALDI. Today, MALDI-TOF is in widespread use in proteomics laboratories.