Proteomics: evolution of the technology.

Abstract

INTRODUCTIONTechnologies enable the advancement of science, which is a process that has been repeated many times throughout the history of science. One of these recent enabling tech-nologies is proteomics. It is an “overnight” sensation that was more than two decades in the making, being developed in a similar time frame as this publication, BioTechniques. Proteomics is the term applied to parallel protein-based analyses. Originally used to describe two-dimensional gel electrophoresis (2-DE) and mass spectrometric analyses of proteins of interest, the term has been applied to any parallel protein analysis method—and even some that really aren’t. This article focuses on parallel protein analyses that result in the identification of components within a system (profil-ing), their posttranslational modifications and/or quantitative analyses of the amounts of the proteins, and modifications or activities, rather than other fields that come under the banner of proteomics, such as structural biology methods that have been reviewed elsewhere (1). We will address how these tech-nologies have impacted science to date and how they might have an impact in the future.WHERE HAVE WE COME FROM?To get to where we are now, a quick glimpse at the past with a couple of limited examples is warranted. Note that this perspective is not intended as a comprehensive review, so only a few pertinent references will be cited per section. There are several recent review articles in the field that pro-vide more in-depth coverage (2,3). Reports of 2-DE, as we know it today, were published in 1975 by three groups and provided the first glimpse at the complexity of the protein content of cells and, as such, can be thought of as marking the true beginning of proteomics (4–6), almost 20 years before the term “proteomics” was coined. It is still the highest resolution protein separation method avail-able and provided the first means of interrogating the levels of hundreds of proteins and their isoforms. However, in 1975, the ability to identify the proteins that displayed an interest-ing regulation proved difficult. Determining the relative in-tensity of the hundreds of spots between numerous gels was also problematic (as was the reproducibility of the method). Automated protein sequencers that could analyze the amount of protein in 2-DE spots had not been developed at that time (that occurred during the following decade), algorithms for image analysis were at an early stage, and computers that could handle the large amounts of data that multiple high-resolution images contained did not yet exist. Several prob-lems had to be solved before this technology could provide answers to detailed biological questions. However, even in its nascent form, discoveries were made, such as the oscillating levels of proliferating cell nuclear antigens (PCNAs) during the cell cycle observed from cell biology experiments in the 1980s (7,8). Protein-DNA interaction studies also revealed the fos-jun interaction (9). However, the full impact of the technology was still limited because it was mostly conducted in the laboratories of a relatively few practitioners of the art. For that to change, a number of advances had to be made, in-cluding some indirectly associated with the field.The advances that enabled proteomics can be thought of as 4-fold (Figure 1). (i) Image analysis methods needed to improve, as did the reproducibility of the 2-DE method, both of which continue to this day. (ii) A method for the sensitive identification of gel-separated proteins was required that ad-vances in mass spectrometry (MS) provided in the late 1980s. (iii) The content of the sequence databases exploded initially with the large-scale expressed sequence tag (EST) sequenc-ing efforts in both the public and private domains and sub-sequently in genomic sequencing efforts. (iv) Computational methods for the correlation of the results of mass spectro-metric analyses with the content of sequence databases were developed. These advances all occurred in the early 1990s, and in 1994, at the first 2-DE meeting in Siena, Italy, the term “proteome” was coined, short for the “protein complement of the genome” (10). The time was right to spark renewed inter-est in this field, which had been so promising for so many years, but had yet to enter the mainstream of biological analy-ses. During the 1990s, the realization of the genomics revolu-tion was taking place, and the potential for true global-scale analyses was becoming a reality with methods such as yeast two-hybrid analysis coming to the fore, and the improvement of EST and genomic sequencing efforts (11).Before the introduction of these technologies, reduction-ist approaches were often employed in the pursuit of the individual proteins or protein families that contributed to an activity/pathway of interest in hypothesis-driven research