FD-LC-MS/MS Method for Determining Protein Expression and Elucidating Biochemical Events in Tissues and Cells

Abstract

To study biochemical events in tissues and cells, we have developed a novel proteomics approach, FD-LC-MS/MS, which consists of fluorogenic derivatization (FD), LC 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-MS/MS. Fluorogenic reagents such as 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulphonamide (DAABD-Cl) were designed to have high sensitivity for HPLC-fluorescence and -MS/MS detection and reactivity for cysteine residues in proteins. This comprehensive differential proteomics approach was applied to several tissues, such as mouse liver, mouse brain, horse muscle, breast cancer cell lines, and mouse heart, in order to study fluctuations in protein levels in tissues and cells.