Development and application of FD-LC-MS/MS proteomics analysis revealing protein expression and biochemical events in tissues and cells

Abstract

It is routine to search for and recognized genetic defects in human disorders to provide knowledge for diagnosis, treatment, and protection against diseases. It is also important to investigate and demonstrate the cause of a disease from the proteomic perspective, because intracellular signaling systems depend on protein dynamics. Demonstrating changes in protein levels enables us to understand biochemical events during the initiation and progression of a disease. To understand changes in protein levels in tissues and cells, we have developed a novel proteomics approach, FD-LC-MS/ MS. This 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 HPLC and tandem mass spectrometry (MS/MS) with a database-searching algorithm. The method is highly sensitive (femtomole-level detection) through the use of less noisy fluorogenic rather than fluorescence derivatization, and en- ables precise and comprehensive relative quantitation of protein levels (between-day relative standard deviation of peak heights of ca. 20％) by combining FD with HPLC separation. In this paper, after a simple review of differential profiling using FD-LC-MS/MS, for example the analysis of stimulated vs. unstimulated samples, we introduce the development and application of the FD-LC-MS/MS method for comprehensive differential proteomics of several tissues, including mouse liver, mouse brain, and breast cancer cell lines, to reveal protein levels and biochemical events in tissues and cells.