1. Introduction
Intense efforts over the last few years have resulted in the availability, at the time of writing (February 2005), of complete genome sequences for 256 organisms (21 archael, 203 bacterial, 32 eukaryotic), including man. This wealth of information is an invaluable resource that will allow comprehensive studies of gene expression that will in turn lead to new insights into cellular functions that determine biologically relevant phenotypes in health and disease. The understanding that one gene can encode more than a single protein has led to a realization that the functional complexity of an organism far exceeds that indicated by its genome sequence alone. While powerful techniques such as DNA microarrays and serial analysis of gene expression (SAGE) make it possible to undertake rapid, global transcriptomic profiling of mRNA expression, processes including alternative mRNA splicing, RNA editing, and co- and posttranslational protein modification make it essential to undertake expression studies at the protein level. The concept of mapping the human complement of protein expression was first proposed more than 25 years ago (Anderson and Anderson, 1982), with the development of a technique in which large numbers of proteins could be separated simultaneously by two-dimensional polyacrylamide gel electrophoresis (2-DE) (O’Farrell, 1975). The term “proteome” was not established until the mid-1990s (Wasinger et al., 1995) when it was proposed to define the protein complement of a genome. This article will give an introduction to expression profiling in which the individual proteins in a complex sample are separated prior to semiquantitative analysis, and then identified usually using techniques of mass spectrometry. For an introduction to alternative strategies for expression profiling of unresolved complex protein samples, see Article 21, Separation-independent approaches for protein expression profiling, Volume 5.
2. Two-dimensional gel electrophoresis (2-DE)
The technique of two-dimensional gel electrophoresis (2-DE) in which proteins are separated in the first dimension according to their charge properties (isoelectric point, pi) under denaturing conditions, followed by their separation in the second dimension according to their relative molecular mass (Mr) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was developed more than 25 years ago (O’Farrell, 1975). Nevertheless, it remains the core technology of choice for the majority of applied proteomic projects (Gorg et al., 2004; see also Article 22, Two-dimensional gel electrophoresis, Volume 5 and Article 29, Two-dimensional gel electrophoresis (2-DE), Volume 5) due to its ability to separate simultaneously thousands of proteins and to indicate posttranslational modifications that result in alterations in protein pi and/or Mr. Large-format (24 x 21cm) 2-D gels can routinely separate around 2000 protein spots. Moreover, recent developments including the use of narrow range “zoom” gels (see Article 22, Two-dimensional gel electrophoresis, Volume 5 and Article 29, Two-dimensional gel electrophoresis (2-DE), Volume 5) and fluorescent dyes that facilitate the multiplex analysis of samples (see Article 25, 2D DIGE, Volume 5 and Article 30, 2-D Difference Gel Electrophoresis – an accurate quantitative method for protein analysis, Volume 5) make it possible to achieve greater proteomic coverage combined with more accurate differential expression analysis. Additional advantages of 2-DE are the high-sensitivity visualization of the resulting 2-D separations (see Article 27, Detecting protein posttranslational modifications using small molecule probes and multiwavelength imaging devices, Volume 5), compatibility with quantitative computer analysis to detect differentially regulated proteins (Dowsey et al., 2003; see also Article 26, Image analysis, Volume 5), and the relative ease with which proteins from 2-D gels can be identified and characterized by mass spectrometry (see Article 31, MS-based methods for identification of 2-DE-resolved proteins, Volume 5).
3. Alternatives to 2-DE
Despite the many advantages of 2-DE, there are alternative protein separation strategies. Perhaps the simplest alternative to 2-DE is the use of one-dimensional SDS-PAGE to separate proteins in the sample, on the basis of their Mr followed by protein identification by tandem mass spectrometry (MS/MS), such that several proteins comigrating in a single band can be identified. This method is limited by the complexity of the protein mixture that can be analyzed but is well suited for the analysis of membrane proteins, and has also been successfully applied to the study of protein complexes (Figeys et al., 2001). Other approaches avoid the use of gels altogether by combining liquid chromatography (LC) and MS. In these so-called shotgun approaches, a tryptic digest of the sample is separated by one or more dimensions (typically ion-exchange combined with reverse-phase) of LC to reduce the complexity of peptide fractions. These are subsequently introduced (either on-or off-line) into a tandem mass spectrometer for sequence-based identification. For example, the so-called MudPIT approach (Wolters et al., 2001) identified around 1500 yeast proteins in a single analysis (Washburn et al., 2001). An alternative to this approach that is more robust than multidimensional chromatography, while still allowing complex samples to be analyzed, has been termed GeLC -MS/MS (Schirle etal., 2003). Here, tryptic digests of protein bands excised from the SDS-PAGE gel are separated by one-dimensional RP-HPLC prior to on- or off-line MS/MS analysis. However, a major limitation of such approaches is that unless combined with some form of stable isotope labeling or “mass tagging”, they provide no information on semiquantitative abundance of proteins and are very limited in their ability to detect posttranslational modifications.
The former problem is currently being addressed by the development of a range of MS-based techniques in which stable isotopes are used to differentiate between two or more populations of proteins (Han et al., 2001). In general, this approach consists of four steps: (1) differential isotopic labeling of the two (or more) protein mixtures, (2) digestion of the combined labeled samples with a protease such as trypsin or Lys-C, (3) separation of the peptides by multidimensional LC, and (4) semiquantitative analysis and identification of the peptides by MS/MS. Currently, the most widely used method is the isotope-coded affinity tag (ICAT) (Han etal., 2001; see also Article 23, ICAT and other labeling strategies for semiquantitative LC-based expression profiling, Volume 5), but there are a variety of other approaches involving labeling with stable isotopes at the whole cell, intact protein, or tryptic peptide level (Julka and Regnier, 2004; Ross et al., 2004; see also Article 23, ICAT and other labeling strategies for semiquantitative LC-based expression profiling, Volume 5). Although these approaches are promising, there are caveats: (1) their quantitative reproducibility needs to be established, (2) the dynamic range of the these techniques may be little better than 2-DE, and (3) there is evidence that they can be complementary to a 2-DE approach in identifying a different subset of proteins from a given sample (Kubota et al., 2003).
4. Conclusion
The current array of proteomic techniques makes it possible to characterize global alterations in protein expression associated with the progression of many different biological processes, including human disease. However, there is still no one method that is suitable for the analysis of all samples, and for many projects it is likely that a combination of proteomic platforms, both gel and nongel based, will have to be applied to provide the required depth of proteomic coverage.
