Isotope-Coded Affinity Tagging Technique Using Avidin Functional Affinity Electrophoresis: An Alternative to an Avidin Affinity Column

Abstract

Avidinfunctionalaffinityelectrophoresis(AFAEP)issubstitutedforanavidinaffinitycolumn(AAC) to capture biotinylated peptides in the Isotope-Coded Affinity Tagging (ICAT) technique which is a valuable tool in quantitative proteomics. In this new technique, the AFAEP-captured ICAT-labeled biotinylated peptides are extracted with the biotin tag intact from the polyacrylamide gel piece with aqueous 95% formamide (pH 8.2) at 65C for 20 min, and then detected by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Bovine serum albumin (BSA) and the 12 C- and 13 C-ICAT reagents are used to test this AFAEP-ICAT technique. The results show that both AFAEP and AAC methods providequantitativeinformationoftherelativeamountsof 12 C-and 13 C-ICAT-labeledbiotinylatedtryptic peptides of BSA in a sample. Compared with AAC, the AFAEP is cheaper to perform, more stringent in capturing the biotinylated peptides, and capable of simultaneously processing multiple samples.