New Atomic Mass Tags for Enhanced Multiplexing Capability of Multiplexed Ion Beam Imaging Time-of-Flight (MIBI-TOF)...

Antibodies conjugated to metal chelating polymers are routinely used in high-dimensional multiplexed single cell mass spectrometric imaging techniques, such as immunohistochemistry-based multiplexed ion beam imaging by time-of-flight (MIBI-TOF) mass spectrometry, imaging mass cytometry (IMC), and flow cytometry-based CyTOF. However, successful multiplexed capability of these techniques is heavily dependent on the stability of the metal-chelates used. Chelate stability is governed by the ionic radius of the metal used, which in some cases can fall below or exceed the optimal range for commercially available DTPA-based polymers. In this study, we have developed and optimized macrocyclic chelators for metals with relatively small (i.e., Ga) or large (i.e., Tl) atomic radii. In agreement with previously published studies, we observed NOTA to be a suitable chelator for Ga, whereas DOTA was found to be an ideal chelator for Tl and larger lanthanides, such as La, Ce, and Pr. DOTA and DTPA chelator dendrimers were synthesized and conjugated to primary antibodies that were subsequently used for tissue staining. Antibodies conjugated with the DOTA-dendrimer were more stable and exhibited more specific staining than those modified with the corresponding DTPA-dendrimer. With these new chelates, we incorporated seven new reporter channels into a highly multiplexed MIBI-TOF imaging study containing 44 protein epitope markers on various tissues. To the best of our knowledge, this is the largest multiplexed panel used to date for MIBI-TOF applications.