Two dimensional electrophoresis
There is now a challenge to replace gels with alternative methods of expression analysis. The most promising method is two-dimensional chromatography of whole cell protein digests coupled to mass spectrometry.
The method has been developed by John Yates and termed Multi-dimensional Protein Identification Technology. Despite the large number of peptides involved, compared to a 2D-PAGE bases approach the actual level of complexity is greatly reduced. Since a single human gene will exist on average in ten different protein forms due to splicing or post-translational modification, the digestion of the proteins reduces 10 proteins to 50 peptides plus 10-20 modified ones instead of 10x50 after protein separation.
The complexity of the problem can be further reduced by selective affinity capture of say cysteine containing peptides using an isotopic label to enable quantification. The Isotopically-coded affinity tagging approach, called ICAT, was developed by the Ruedi Aebersolds laboratory approach in combination with MudPIT is rapidly gaining popularity and is outlined below. Thanks to Ruedi Aebersold for the figures.
The Isotope Coded Affinity Tagging strategy for quantifying differential protein expression.
The ICAT method
The structures of the original ICAT reagents are shown in Figure (a). The reagent consists of three moieties: an affinity tag (biotin), which is used to isolate ICAT-labelled peptides; a linker that can incorporates stable isotopes and a maleimide group which reacts specifically with the thiol group of cysteine. Two labelled forms of the reagent are used, the heavy containing eight deuteriums and the light with none. Figure (b) shows a schematic diagram of the ICAT approach. Proteins from two different cell states are extracted and labelled with the light or heavy ICAT reagents. The samples are then combined and digested. The ICAT-labelled peptides are isolated by affinity chromatography using an avidin column and then analysed HPLC-MS (/MS) directly or by MALDI of the collected HPLC fractions. The ratio of the peaks areas for specific ICAT-labelled pairs defines the relative abundance of its parent proteins between the two cell states (see the figure below).
Isotopic-labelling Strategy (ICAT)
Limitations of the methods
Despite the debates raging about the relative merits of the protein and peptide-based approaches, all share the same limitations. The dynamic range of protein expression in a cell is around 107 and in fluids such a blood serum it is probably around 1012. The two main detection methods used, mass spectrometry and fluorescence can only deal with four orders of magnitude. Thus there is no tool, chip, gel or HPLC that can cover the range of protein expression found in nature. Secondly, all of the techniques are dependant on protein digestion to get fragments for identification or analysis of post-translational modifications (PTMs) and all proteases cease to be effective at substrate concentrations at the nanomolar level. Even antibody chips must be analysed to see if the fluorescence is due to the antigen protein or to cross-reactivity or even the binding of the protein antigen plus a series of other proteins in a complex since the binding is done under non-denaturing conditions. Thus whichever approach one takes one will only see a subset of the proteins that are present in the cell or serum.