Isotope-coded affinity tagging technique using avidin functional affinity electrophoresis: An alternative to an avidin affinity column

Avidin functional affinity electrophoresis (AFAEP) is substituted for an avidin affinity column (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 65 °C for 20 min, and then detected by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Bovine serum albumin (BSA) and the C- and C-ICAT reagents are used to test this AFAEP-ICAT technique. The results show that both AFAEP and AAC methods provide quantitative information of the relative amounts of C- and C-ICAT-labeled biotinylated tryptic 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.