Towards an Integrated Analytical Technology for the Generation of Multidimensional Protein Expression Maps

Ruedi Aebersold*, Daniel Figeys, Steven Gygi, Garry Corthals, Paul Haynes, Beate Rist, Jamie Sherman, Yanni Zhang, David Goodlett

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)


One of the most dramatic current developments in bio-logical research is the shift from the analysis of single genes and proteins to the comprehensive analysis of bi-ological systems and pathways. This shift is at least in part the consequence of the development of automated, high-throughput genomic technologies which have advanced to a point where, in principle, it has become pos-sible to determine complete genome sequences and to quantitatively measure the mRN A levels of all the genes expressed in a cell. Currently, no comparably powerful technology is available for the analysis of biological sys-tems on the protein level. Proteins are, however, the most significant class of biological effector molecules and a complete model of a biological process cannot be established without knowledge of the identity, function, and state of expression and activity of the proteins in-volved. A quantitative expression map of the proteins in a cell or tissue has been termed a proteome. We will describe a suite of technologies for the identification and analysis of the proteins which constitute a biological sys-tem, i.e., for the description of proteomes. Mass-Spectrometry Based Identification of Proteins Numerous techniques for the identification of pro-teins have been described and successfully employed. They include N-terminal and internal protein sequenc-ing, identification by amino acid composition analysis, and a variety of mass spectrometric (MS) approaches. Currently, MS or tandem mass spectrometric (MS/MS) analysis of a protein digest and correlation of the obtained data with sequence database entries is considered the method of choice for protein identification. The sensitivity and the throughput of these techniques are in-versely related. The most sensitive techniques such as solid-phase extraction capillary electrophoresis MS/MS and nanospray MS/MS have been difficult to automate. Techniques of lower sensitivity such as liquid chroma-tography MS/MS have successfully been automated for higher sample throughput. To achieve both high sensi-tivity and automation in the same system, we have used photolithography/etching techniques to develop micro-machined devices which can be connected on-line with electrospray ionization (ESI) MS/MS. Multiple samples concurrently present in different reservoirs on the device are sequentially and automatically mobilized by an array of high-voltage relays and electroosmotically pumped, either directly or after Chromatographie or electrophor-etic separation, to the ion source of the MS instrument. We have used this system for the identification of nu-merous proteins separated by high-resolution two-dimensional gel electophoresis. Analysis of the State of Protein Modification Frequently, the state of modification of a protein and the specific constellation of modified proteins in a cell indicate the state of activity of the protein and the cell, respectively. The analysis of protein modifications is complicated by a frequently low stoichiometry and complex patterns of modification, low abundance of the modified proteins, and the large number and chemical diversity of protein modifications. The task of analyzing the state of protein modification in biological systems has at least three stages. The first is the detec-tion/visualization of the modified proteins in a protein mixture. The second step is the identification of those proteins in a protein mixture which are modified by a specific modification. The third step is the localization of the modification(s) within the polypeptide chain of the identified proteins. Protein and peptide MS and MS/MS are rapidly becoming the methods of choice for the identification as well as the analysis of posttransla-tional modifications. We have developed and successfully applied a suite of techniques for the analysis of the phosphorylation state of proteins. Reversible phosphorylation of proteins at specific sites is an essentially universal mechanism for the control of the activity of the phosphorylated protein and of biological processes. The objectives of the techniques developed were the quantitative determination of the state of phosphorylation of the small amounts of phosphoprotein which can be isolated from cells or tis-sues representing a specific state. These objectives were achieved by a combination of biochemical, enzymatic, Chromatographie, and electrophoretic techniques which were used together with ESI MS/MS. The identification of the proteins in a complex pro-tein mixture which are modified by a specific type of modification is almost universally achieved by metabolic radiolabeling of the protein sample with a metabolic precursor specific for the modification under investigation, followed by the separation of the proteins mixture by high-resolution gel electrophoresis and detection of the modified protein(s) by autoradiography, fluorography, or storage phosphorimaging. This technique is purely de-scriptive and does not in general yield the identity of the modified proteins. We will describe novel approaches based on ESI-MS/MS which, in the same operation, detect the proteins modified by a specific group in complex protein mixtures and also identify the protein by its amino acid sequence. Construction of Multidimensional Protein Expression Maps With current technology, global and quantitative mRNA transcript expression maps can be established rapidly. The construction of similarly comprehensive protein expression maps (proteome maps) is much more labor-intensive and much slower. The added cost, time, and effort required to establish proteome maps is justified by the added information regarding the state of a biological system which can be obtained from proteome analysis. We have correlated the quantitative mRNA transcript expression map obtained from the yeast Saccharomyces cerevisiae with a quantitative proteome map from the same strain of yeast grown under identical conditions. The quantitative mRNA expression data were calculated from SAGE frequency tables described in the literature (Velculescu et al., 1997). The protein expression data were derived from the quantitative and mass spectrometric analysis of homogeneous protein spots in two-dimensional gels. The gels were generated by separating total yeast cell lysates metabolically radiolabeled to equilibrium. The analysis of the results indicated that (i) the mRNA transcript and protein expression levels for specific genes are poorly correlated, (ii) proteins coded for by the same gene frequently migrated to different positions in the two-dimensional protein pattern, indicating the activity of posttranslational modification and processing mechanisms, and (iii) without selective enrichment for low-abundance proteins, current proteome technology is limited to the analysis of the most highly expressed proteins in a cell and is therefore not comprehensive. We therefore conclude that efforts in proteome analysis should be focused on the determina-tion of those parameters describing the state of a biological system which cannot be determined by genomic or genetic means. Such parameters include the quantity of protein expression, protein half-life, the state of modification of individual proteins, and the state of association of proteins with other molecules. Protein analysis technology is in the process of being transformed from a technology focusing on the analysis of single proteins to the analysis of all the proteins which constitute a biological system. MS and MS/MS have become the methods of choice for both the identification of proteins in biological systems and the analysis of their covalent structure. The limitations inherentin current protein analytical technologies with respect to sensitivity and sample throughput suggest that protein analysis is best practiced in conjunction with genomic analysis and that protein analysis should focus on the determination of those parameters characterizing biological systems which can only be determined by direct protein analysis.

Original languageEnglish
Pages (from-to)533
Number of pages1
JournalProtein Journal
Issue number6
Publication statusPublished - 1998
Externally publishedYes


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