In-situ monitoring of protein labeling reactions by matrix-assisted laser desorption/ionization mass spectrometry.
Taking the labeling reaction of horse heart cytochrome c or ubiquitin with biotinamidocaproate N-hydroxysucchinimide ester (biotin-NHS) as test cases, this report demonstrates the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for in-situ monitoring of the labeling process and for determining the composition of the labeled products without the need for prior separation. The effects of pH and starting materials concentration on the labeling process were investigated in detail. Our MALDI MS results show that: (1) labeled products are always mixtures of different conjugates, which may explain peak broadening found in chromatographic studies of labeling reactions; (2) the higher conjugate fractions become more prominent as the labeling reaction proceeds, with a concomitant exponential decline of the lower conjugate fractions; (3) biotin-NHS can be incorporated into peptides and protein in a stepwise and controlled manner simply by adjusting the molar ratio of the starting materials.
Lu
J
J
Department of Chemistry, ETH Zentrum, Zürich, Switzerland.
Zenobi
R
R
D000614:Aminocaproates / Q000737:chemistry*; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry*; D006321:Heart / Q000502:physiology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D014452:Ubiquitins / Q000737:chemistry*
D000614:Aminocaproates; D003574:Cytochrome c Group; D014452:Ubiquitins; D001710:Biotin; C048592:biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester