Use of protein chip mass spectrometry to monitor biotinylation reactions.
Surface-enhanced laser desorption/ionization time-of-flight analysis was used to monitor both the kinetics and heterogeneity of product formation during the biotinylation of a number of model proteins and peptide targets. The selected molecules were the IgG-binding protein, protein A, human serum albumin, and a synthetic peptide corresponding to the N terminus of a streptococcal M1 protein. The extent of biotinylation was determined by kinetic analysis of the shift in molecular mass from the native material. Each residue modified by reaction with N-hydroxysuccinimide biotin resulted in an addition of approximately 341 amu to the native protein or polypeptide. The novelty of the method was in the ability to determine the molecular mass shift, without first separating the targeted molecule from the biotinylating reagent. The analysis was rapid, simple, and provided information on the average number of biotin molecules added and the homogeneity of the resulting product.
Blazer
Levi L
LL
Department of Biology, Juniata College, 1700 Moore Street, Huntingdon, PA, 16652, USA.
Boyle
Michael D P
MD
D000942:Antigens, Bacterial / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D019921:Biotinylation; D002352:Carrier Proteins / Q000378:metabolism*; D007700:Kinetics; D040081:Protein Array Analysis; D012709:Serum Albumin / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013205:Staphylococcal Protein A / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D012709:Serum Albumin; D013205:Staphylococcal Protein A; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; C014689:streptococcal M protein; D001710:Biotin