Target proteins identification

Target Protein Identification of HBC, an Inhibitor of Cell-Cycle Progression... 

Cheng K, Sloan A, McCorrister S, et al. Fit-for-purpose curated database application in mass spectrometry-based targeted protein identification and validation. BMC Res Notes. 2014 7 444. doi 10.1186/1756-0500-7-444. 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Although a number of cell lines were shown to be sensitive to inhibition by PatA (Low et al., 2005), we selected RKO cells (IC50 of 0.4 nM in cell proliferation assay) to prepare lysates for the isolation and identification of target protein(s). We often select RKO cells for target identification of small molecules using biotin-conjugates, because they appear to be particularly suitable for target protein isolation. 

Mesange F, Sebbar M, Capdevielle J, Guillemot JC, Ferrara P, Bayard F, Poirot M, Faye JC (2002) Identification of two tamoxifen target proteins by photolabeling with 4-(2-morpholinoethoxy)benzophenone. Bioconjug Chem 13(4) 766-772... 

The result of a standard photoaffinity experiment can provide an output in three levels [8] as shown in Scheme 2. The photolytically induced targeted introduction of a (radio )label onto the target biopolymer helps to identify any specific binding proteins (first level of identification) responsible for a particular action in the signaling cascade or to identify the binding domain within the target protein (second level of identification) or allows one to identify the amino acid sequence of the binding protein (third level of identification). 

Vijayasaradhi, S., Xu, Y., Bouchard, B., and Houghton, A. (1995). Intracellular sorting and targeting of melanosomal membrane proteins identification of signals for sorting of the human brown locus protein, gp75. J. Cell Biol. 130, 807-820. 

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