Determination of the stoichiometry of protein complexes using liquid chromatography with fluorescence and mass spectrometric detection of fluorescently labeled proteolytic peptides.
A method for the determination of the stoichiometry of protein complexes has been developed, which is based on proteolytic digestion of the complex, labeling with a fluorescent reagent, specific for amino or sulfhydryl groups, and separation by liquid chromatography with fluorescence and mass spectrometric detection. The intensity of the fluorescence signal of the labeled peptides resulting from different proteins is directly proportional to the stoichiometry of these proteins in the complex. The performance of the method was evaluated with standard peptides and proteins to ensure that accurate molar ratios can be obtained from the fluorescence chromatogram. Standard deviations of the measured molar ratio from the expected molar ratio were below 10% for both peptides and proteins. The method was finally employed for the determination of the stoichiometry of the 1:1 complex of sFc gamma RIII and hFc1. Using the described methodology, a stoichiometry of 1:1.1 was measured, which agrees well with a 1:1 complex.
Hochleitner
Elisabeth O
EO
Analytical Protein Chemistry Group, Max Planck Institute of Biochemistry, Martinsried, Germany. hochleit@biochem.mpg.edu
Sondermann
Peter
P
Lottspeich
Friedrich
F
D000809:Angiotensins / Q000737:chemistry; D001920:Bradykinin / Q000737:chemistry; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D000809:Angiotensins; D005452:Fluoresceins; D008024:Ligands; D010455:Peptides; C015296:5-iodoacetamidofluorescein; D001920:Bradykinin