An identification method for altered proteins in tissues utilizing fluorescence derivatization, liquid chromatography, tandem mass spectrometry, and a database-searching algorithm.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now widely used as a tool for proteomic studies. For the sensitive determination of proteins in 2D-PAGE, fluorescence derivatization of primary amino moieties of proteins with cyanine dyes was recently developed. However, precipitation of the proteins could occur if completely derivatized because of the lower solubility of the resultant derivatives owing to the hydrophobicity of the reagents and the loss of the hydrophilic primary amino moieties. Thus, in this paper, a water-soluble and thiol-specific fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, was adopted for the derivatization of proteins in tissues either with and without stimulation. Then, the method follows a separation of the derivatives by liquid chromatography with fluorescence detection, an isolation of only the altered proteins, an enzymatic digestion of the isolated proteins, and an identification of the proteins by liquid chromatography/MS/MS with the database-searching algorithm. By using this method, we identified the altered expressions of five increased proteins (e.g., pancreatic polypeptide) as well as three decreased proteins (e.g., insulin 2) in the islets of Langerhans in Wistar rats 2 days after they were subcutaneously administered with dexamethasone.
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Chifuyu
C
Toriumi
Kazuhiro
K
Imai
D000465:Algorithms; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D030562:Databases, Protein; D003907:Dexamethasone / Q000008:administration & dosage / Q000494:pharmacology; D005453:Fluorescence; D005786:Gene Expression Regulation / Q000187:drug effects; D057927:Hydrophobic and Hydrophilic Interactions; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D012995:Solubility
D010455:Peptides; D011506:Proteins; D012710:Serum Albumin, Bovine; D003907:Dexamethasone