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 desorptio n/ionization (MALDI) mass spectrometry for insitu monitoring of the labelin g process and for determining the composition of the labeled products witho ut the need for prior separation. The effects of pH and starting materials concentration on the labeling process were investigated in detail. Our MALD I MS results show that: (1) labeled products are always mixtures of differe nt conjugates, which may explain peak broadening found in chromatographic s tudies of labeling reactions; (2) the higher conjugate fractions become mor e prominent as the labeling reaction proceeds, with a concomitant exponenti al decline of the lower conjugate fractions; (3) biotin-NHS can be incorpor ated into peptides and protein in a stepwise and controlled manner simply b y adjusting the molar ratio of the starting materials.