Emerging strategies in mass-spectrometry based proteomics.

Abstract

The introduction of peptide mass spectrometry and the progress in the various cDNA and genome sequencing projects made the identification of proteins faster and more efficient than ever before. When mass spectrometry and bioinformatics were combined with the high-resolution two-dimensional gel electrophoretical separation of complex protein mixtures, the large-scale identification of proteins in a single experiment became feasible. Based on this analytical potential, the concept of ‘proteomics’ was developed as a systematic approach for the global analysis of all proteins present in a given experimental system under investigation, representing a snapshot of the actual state of the system. The expectation was that the comparison of several such snapshots obtained from the same experimental system in different states might reveal the molecular bases of many diseases. The term ‘proteome’ thereby was coined in analogy to the term ‘genome’, which refers to the whole entity of genes in an organism. The analogy is also mirrored in the expectations of what would be observed when there are dynamic changes in the system being investigated. This point of view dominated the experimental set-ups that I call here the first generation of proteomics studies; similar to the appearance or disappearance of new mRNA species as assessed by the differential display method or by using cDNA arrays, there was an expectation that changes in the proteome would show up as difference spots on two-dimensional gels after separation of crude and complex protein samples. The appearance of difference spots could be due to new synthesis of proteins, protein degradation, or post-translational modifications. Thus, the methodology was suited to detecting dynamic changes in the experimental system that were not accessible at the level of the mRNA/cDNA, and this allowed an analysis at a higher level of complexity as compared to genomics and transcriptomics. However, the proteins that showed up as spots on gels were treated as isolated entities. Apart from the technical limitations of the approach, there are biochemical mechanisms underlying dynamic changes of the proteome that cannot be addressed by this strategy: these comprise mechanisms that are independent of protein synthesis or degradation. Among these mechanisms, there are protein translocations, altered protein–protein interactions or altered catalytic activity of enzymes giving rise, for example, to altered metabolite formation. The need to cover these aspects of dynamic changes in the proteome recently led to the design of a new generation of proteome analysis strategies that consider protein interactions, the subcellular distribution of proteins, or the metabolic profile of the system under investigation. These strategies incorporate new methodological tools and well-established biochemical methods. However, they are still true proteomics strategies because of their comprehensive scope, in contrast with the application of protein chemical methods to the characterization of, say, single protein components in other cell biological or biochemical studies. The three minireviews in this series present proteomics strategies designed to address protein-protein interactions, subcellular distribution of proteins, and catalytic activity of proteins as assessed by analyzing the metabolome and the proteome in an integrated approach. First, Bauer and Küster give an overview of techniques that are suited for the analysis of protein complexes by protein chemical means. In particular they elaborate on the analysis of multiple protein complexes using the tandem affinity purification (TAP)-tag technique which they used in a recent breakthrough study on the large-scale identification of multiprotein complexes in yeast. Secondly, Fiehn and Weckwerth give an overview of experimental approaches to assess the metabolome and the proteome of the experimental system investigated at a qualitative and quantitative level. They elaborate on the concept of metabolic networks that may be deduced from systematic metabolomics studies. And thirdly, Dreger gives an overview of recent studies aimed at the description of partial proteomes at the level of subcellular structures. Rather than replacing the classic ‘first-generation’ proteomics approach, the now emerging strategies of ‘second-generation’ proteomics contribute valuable tools to the growing repertoire of proteome analysis strategies. The ultimate goal is the analytical capability to address all levels of protein structure and function in comprehensive studies to describe the experimental system under investigation in terms of the concept of Systems Biology.