Site-Specific Fluorescent Labeling of Hemagglutinin-Specific Antigen Binding Fragment through Amine Chemistry Revealed by Mass Spectrometry

To capture the structure of assembled hemagglutiantigen binding fragments (Fabs) were labeled by fluorescent (FLR) dyes as probes to highlight the HA trimers displayed on the assembled tetravalent HA nanoparticles for a qualitative localization microscopic study. The FLR dyes were conjugated to the Fabs through N-hydroxysuccinimide (NHS) ester mediated amine coupling chemistry. The labeling profile, including labeling ratio, distribution, and site-specific labeling occupancy, can affect the imaging results and introduce inconsistency. To evaluate the labeling profile so as to evaluate the labeling efficiency, a combination of intact mass measurement by MALDI-MS and peptide mapping through LC-MS/MS was implemented. At the intact molecular level, the labeling ratio and distribution were determined. Through peptide mapping, the labeled residues were identified and the corresponding site-specific labeling occupancy was measured. A systematic comparative investigation of four different FLR-labeled 1H01-Fabs (generated from H1 strain HA specific mAb 1H01) allowed accurate profiling of the labeling pattern. The data indicate that the labeling was site-specific and semiquantitative. This warrants the consistency of single-particle fluorescent imaging experiments and allows a further imaging characterization of the single nanoparticles.