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Mass spectrometric detection of biotinylated peptides captured by avidin functional affinity electrophoresis
Author(s) -
Lee BaoShiang,
Krisnanchettiar Sangeeth,
Lateef Syed Salman,
Gupta Shalini
Publication year - 2005
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1865
Subject(s) - chemistry , biotinylation , avidin , chromatography , affinity electrophoresis , affinity chromatography , biochemistry , enzyme
Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry was used to detect biotinylated peptides captured by avidin functional affinity electrophoresis (AFAEP). Peptide samples loaded onto AFAEP were heated with sodium dodecyl sulfate to ensure that the peptides are negatively charged, and thus migrate electrophoretically toward the cathode through the embedded avidin zone in the middle of the gel. To detect the biotinylated peptides, the band containing the avidin‐biotinylated peptide complexes was excised from a 7.5% w/v native polyacrylamide gel, and biotinylated peptides were extracted with aqueous 95% v/v formamide (pH 8.2), aqueous 6 M guanidine HCl (pH 1.5), or water, at temperatures from 4 to 95°C for periods from 5 min to 24 h. It was observed that all three solvents are capable of extracting biotinylated peptides and avidin from the gel, but the best results were obtained with aqueous 95% v/v formamide (pH 8.2) at 65°C for 20 min. However, some AFAEP‐captured biotinylated peptides are not stable and are extensively modified by formamide during extraction at too high a temperature or too long an extraction time. Copyright © 2005 John Wiley & Sons, Ltd.