Development and Application for Comprehensive Quantitative Analysis of Fluorogenic Derivatized Proteins

It is important to search for and demonstrate the defects in genetic disorders in order to provide knowledge for the diagnosis, treatment, and protection against diseases. Proteomics analysis is an option used to search for and to demonstrate the cause of disease from the proteomic perspective, since the intracellular signaling system actually consists of expressed protein dynamics. However, because of the difficulty of efficient separation of high-molecular-weight compounds, such as proteins, high-performance liquid chromatography (HPLC) is not commonly used for the separation of mixtures of proteins in proteomics analysis. We have developed a novel proteomics approach, FD-LC-MS/MS, which consists of fluorogenic derivatization (FD), HPLC separation and detection/quantification of proteins in a biological sample, followed by the isolation and tryptic digestion of target proteins, and then their identification using nano-HPLC-tandem mass spectrometry (MS/MS). This method is highly sensitive due to less noisy fluorogenic rather than fluorescence derivatization, and enables a precise and comprehensive relative quantitation of protein levels (ca. 20% between-day relative standard deviation of peak heights) by combining FD with HPLC separation. In this article, after a simple review of other types of proteomic analyses, we introduce the development and application of the FD-LC-MS/MS method.