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What's FD-LC-MS/MS ?
The FD-LC-MS/MS method consists of fluorogenic derivatization (FD) of proteins using fluorogenic reagents
such as 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl), followed by highly reproducible HPLC separation,
quantification and fractionation of the altered proteins, enzymatic digestion of the isolated proteins, and final identification of the proteins
utilizing HPLC and tandem MS with a database-searching algorithm.
The method is useful for the comprehensive differential proteomics analysis among different stages of cells or tissues,
affording new signaling pathways in cells and finding new biomarkers for cancers.
DAABD-Cl we have synthesized is non-fluorescent itself and reacts with thiol moiety in proteins to give fluorescent proteins.
The fluorescent proteins can be separated and sensitively detected in HPLC with a fluorescence detector.
As shown in the figure, the proteins mixture in sample A are first derivatized with DAABD-Cl,
the derivatized fluorescent proteins are subjected to HPLC-fluorescence detection and the chromatogram A is obtained.
Treating the same procedure for the sample B, the chromatogram B is obtained.
After comparison of the heights of the corresponding peaks between A and B,
the protein with different peak height is isolated and subjected to enzymatic hydrolysis.
The generated peptides mixture is then subject to nano-LC-MS/MS for identification of the subject protein.
The followings are the features of the FD-LC-MS/MS.
・ The usage of the fluorogenic reagents, DAABD-Cl, enables highly sensitive detection of derivatized proteins (10^-15 mol level)
・ The separation by HPLC leads to highly reproducible quantification.
・ The isolation of the sole target protein peak does not lose any amino acid sequence information, such as protein isoforms and post-translational modifications.
・ FD-LC-MS/MS involves a simple set up equipments consisting of a pump, a column, and a fluorescence detector.
The details are given in the following review paper.
・ Imai K, Koshiyama A, Nakata K (2011); Towards clinical proteomics analysis. Biomed Chromatogr 25: 59-64.
・Ichibangase T, Imai K (2012); FD-LC-MS/MS Method for Determining Protein Expression and Elucidating Biochemical Events in Tissues and Cells. Biol Pharm Bull 35: 1393-1400
What's FD-LC-MS/MS? Published papers
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