User Reports
Musashino University, Research Institute of Pharmaceutical Sciences
Japan
Proteome Analysis of Proteins by Fluorescent Derivatization LC-MS/MS Method
The authors have developed a novel comprehensive quantitative proteome analysis method (FD-LC-MS/MS) that differs from conventional shotgun proteomics. This method uses the fluorescent derivatization reagent DAABD-Cl, developed by Professor Emeritus Kazuhiro Imai of the University of Tokyo, and involves the following processes:
- Modification of protein SH groups with DAABD-Cl (FD reaction)
- Comprehensive LC separation and fluorescence detection of derivatized proteins
- Comparison of chromatograms and statistical selection of significant proteins for fractionation
- Enzymatic digestion of fractionated proteins, followed by identification of the original proteins via LC-MS/MS and database matching
This new method offers the following advantages over conventional methods like shotgun proteomics:
- Simple sample pretreatment
(only homogenization and FD reaction) - High sensitivity detection enabled by fluorescent reagents
(minimal risk of losing expressed proteins) - High reproducibility allowing significant difference testing of peak areas (quantitative and differential analysis)
The authors have already achieved significant results using this method in proteome analysis of hepatitis C models, breast cancer cell lines, colon cancer cell lines, and superoxide overexpressing cells.
In this method, column performance is crucial for separating numerous proteins. It has been confirmed that the non-porous ODS column Presto FF-C18, which lacks pores, is superior to porous columns that tend to adsorb proteins within their pores.
The non-porous ODS Presto FF-C18 plays an important role in the precise separation of proteins.
- Imtakt Tecnical Information, TI808E
(Provided by Dr. Tomoko Ichibangase and Dr. Kazuyo Imai, Musashino University, Institute of Pharmaceutical Sciences)
Reference:
Efficient chromatographic separation of intact proteins derivatized with a fluorogenic reagent for proteomics analysis
Tomoko Ichibangase, Itaru Yazawa and Kazuhiro Imai
Biomedical Chromatography, 2013; 27: 1520-1523
発蛍光標識化タンパク質の網羅定量解析法の開発と応用
Tomoko Ichibangase
BUNSEKI KAGAKU, Vol.64 (2015) No.7, p.511-517