Can Proteomics Retire the Western Blot?
J. Proteome Res., 7 (8), 3065, 2008. 10.1021/pr800463v
Proteomics has made great strides toward biologically useful applications, but too often proteomics researchers and biologists still inhabit different worlds. Proteomics as a discipline grew up with the ambitions and pretensions of the genome projects, but biologists mostly want proteomics technology to solve their humdrum analytical problems. The truth is in between, and here I argue that true integration of proteomics technology into molecular biology laboratories could be a paradigm shift for all of biology and biomedicine.
Biologists currently are stuck in a time warp—they basically use the same tools for protein detection as they have for the past 20 years. The combination of 1DE and antibodies as western blotting reagents is the technical basis of entire fields, such as cell signaling. Despite its obvious success and convenience, problems exist with this approach. The strategy is semiquantitative at best, and antibodies, especially those generated against posttranslational modifications, are often of poor specificity. A more fundamental limitation of this method lies in its targeted nature: using antibody detection, biologists can find only what they look for. Unexpected effects in different cellular compartments than the one studied or in different cellular processes go undiscovered. As a result, the cell is not seen as a whole system, and researchers cover the same well-trodden ground over and over again.
Proteomics could solve this problem. Straightforward and gel-free workflows based on high-resolution MS can now cover most cellular proteins in a quantitative experiment (Cell 2007, 130, 395−398). The resulting list of detected proteins also will contain the ones traditionally probed for by western blotting, but they will be measured in a truly quantitative instead of largely qualitative manner. Unlike in traditional biology, however, proteomics results also contain any other changes that have occurred in the cellular proteome. These data will give the researcher the assurance that the investigated effect is one of the major ones. Proteomics also can reveal the modification status of the protein, which currently is seldom probed and, if so, only for specific modifications. In most cases, proteomics will reveal more changes than previously anticipated and provide a wealth of new discoveries and hypotheses to follow.
What do we have to do to make this happen? Basically, we have to make quantitative proteome measurements as accessible and convenient as western blots are now. This may seem like a tall order, but it may not be so for long. First, proteome measurements do not require much material—already quantitative proteomics can be done on the cells in a single Petri dish, as in western blotting. Second, antibody reagents are poor or nonexistent for many proteins, especially the ones that have not been studied for many years. Detecting these proteins is already easier by quantitative MS. Third, when pull-downs or purified fractions have to be quantitatively analyzed, this task is already within reach of routine analysis. Finally, a kind of Moore’s law is in effect in proteomics, drastically enhancing our analytical capabilities every year. Routine and quick analysis of whole proteomes may be closer than we think, especially if we all put our minds to this challenge. Equally important, proteomics should be incorporated into the education of biologists, so that the next generation of biologists will naturally turn to this powerful technology.
The positive effects of such a paradigm shift would be hard to overestimate. This shift would lead to a great quality improvement in contemporary biological research. The transition would also help to make “systems biologists” out of scientists who are currently focusing on all too small a corner of the whole cellular universe.