MALDI mass spectrometry combined with avidin-biotin chemistry for analysis of protein modifications.
A general mass spectrometric method that combines purification and analysis in one step is described for the rapid and sensitive determination of protein modification that involves covalent attachment of a modifying group. In this method, the modifying group is first labeled with a biotin moiety, and the covalent interaction of this group with the targeted protein results in a biotinylated product. The modified protein can then be subjected to enzymatic digestion, followed by the isolation of the biotinylated peptide based on a previously described MALDI method incorporating the avidin-biotin interaction (Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 3382-3387). To illustrate the validity of the method, a study of a model system was undertaken, involving the interaction between avian skeletal muscle troponin C and a sulfhydryl-specific biotinylation reagent. It is shown that isolation of a modified peptide with an immobilized avidin product could be achieved, even in the presence of an excess of contaminating protein. Exoproteases could be added to the crude tryptic digest to generate peptide ladders, each containing biotin, which could be analyzed by the avidin-biotin/MALDI method for sequence information. Complementary sequence information could be obtained from the application of this technique in a tandem sector/time-of-flight mass spectrometer for MALDI MS/MS analysis, which allowed for the identification of the modification site.
Schriemer
D C
DC
Department of Chemistry, University of Alberta, Edmonton, Canada.
Yalcin
T
T
Li
L
L
D000818:Animals; D001360:Avidin / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D002645:Chickens; D020689:Exopeptidases; D018482:Muscle, Skeletal / Q000378:metabolism; D010447:Peptide Hydrolases; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013439:Sulfhydryl Reagents / Q000737:chemistry; D019209:Troponin C / Q000032:analysis; D014357:Trypsin
D010455:Peptides; D011506:Proteins; D013439:Sulfhydryl Reagents; D019209:Troponin C; D001360:Avidin; D001710:Biotin; D020689:Exopeptidases; D010447:Peptide Hydrolases; D014357:Trypsin