Mass spectrometric detection of biotinylated peptides captured by avidin functional affinity electrophoresis.
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 degrees 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 degrees 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.
Lee
Bao-Shiang
BS
Protein Research Laboratory, Research Resources Center, University of Illinois, 835 S. Wolcott Avenue, Chicago, IL 60612, USA. boblee@uic.edu
Krisnanchettiar
Sangeeth
S
Lateef
Syed Salman
SS
Gupta
Shalini
S
D001360:Avidin / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010455:Peptides; D001360:Avidin; D001710:Biotin