| After the discovery that whole cells could be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, much effort has been applied to rapid identification of intact cellular proteins. Several problems, including sample reproducibility, database searching, insufficient mass accuracy, and low resolving power have been noted. These factors have hindered the ability of this technique to rapidly identify proteins. By applying Fourier transform mass spectrometry to analysis of whole cells by MALDI, significant improvements in the previous mentioned factors allow for accurate identifications of individual proteins.; With the implementation of FTMS to intact cell analysis, only a very small fraction of proteins can be identified. Considering the large number of proteins within any proteome, typically several thousands, the question becomes where are the other proteins? One inherent problem with FTMS, when external ionization is used, is that numerous metastable decay products result from analysis. These products cause a problem referred to as chemical noise. To help alleviate this problem, a special growth media doubly-depleted of 13C and 15N is applied to the analysis of E. coli cells. This media dramatically reduces chemical noise present under normal conditions allowing for increases in the number of detectable proteins, their accurate mass measurement and resolving power.; Beyond the application of chemotaxonomy, this method can be applied to the analysis of recombinantly over-expressed proteins. Presently, the primary method to synthesize proteins used in biochemistry is over-expression of a specific protein using microorganisms as the medium for targeted protein production. This is necessary since many proteins of interest do not typically exist in high concentrations naturally. Thus, by allowing microorganisms to over produce a specific protein, the ability to characterize it is notably improved. This method has the potential to optimize the production of a given protein, since during the growth cycle of a microorganism different proteins are expressed at varying rates. All of these methods and techniques demonstrate that MALDI FTMS analysis provides high degrees of mass accuracy and resolving power, over other types of MS detection, that ultimately allow for increased confidence in identifications of specific proteins directly from whole cells. |
