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Isotope‐Coded Affinity Tagging Technique Using Avidin Functional Affinity Electrophoresis: An Alternative to an Avidin Affinity Column
Author(s) -
Lee BaoShiang,
Krishnanchettiar Sangeeth,
Lateef Syed Salman,
Lateef Nabila Salman,
Gupta Shalini
Publication year - 2006
Publication title -
journal of the chinese chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.329
H-Index - 45
eISSN - 2192-6549
pISSN - 0009-4536
DOI - 10.1002/jccs.200600098
Subject(s) - chemistry , biotinylation , avidin , chromatography , affinity chromatography , affinity electrophoresis , bovine serum albumin , matrix assisted laser desorption/ionization , biotin , mass spectrometry , desorption , biochemistry , organic chemistry , adsorption , enzyme
Abstract 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 12 C‐ and 13 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 12 C‐ and 13 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.