Development and Application of Structurally Defined Peptide Oligomeric Tags for Mass Cytometry

To address the issues of high molecular weight polydispersity, poor conjugation uniformity and high steric hindrance of existing polymer or nanoparticle tags for mass cytometry, a structurally defined peptide oligomeric mass tag, 8DOTA-PEG3-Mal, via controlled solid-phase peptide synthesis combined with an iterative block coupling strategy was prepared in this study, and its structure was verified by mass spectrometry. Endowed with low steric hindrance, this tag enabled efficient antibody conjugation. Silver staining of gel electrophoresis showed that 2 and 1 tags were conjugated to heavy and light chains of the labeled antibody, respectively, with each IgG stably loading 48 metal ions, demonstrating excellent conjugation uniformity. Analyses of the staining index and separation index indicated that the CD45 antibody labeled with this tag exhibited high specificity and sensitivity in staining Jurkat/HeLa cells. In the multiparametric detection of peripheral blood mononuclear cells, the tag-labeled antibodies could accurately distinguish lymphocyte subsets, with the proportions of each cell population highly consistent with those detected by the commercial Maxpar X8 reagent tag. The antibody-tag conjugate showed excellent long-term storage stability, and no significant attenuation of the detection signal was observed after 120 days of storage at 4 ℃. In conclusion, the 8DOTA-PEG3-Mal tag integrated excellent metal chelating ability, low steric hindrance and high antibody conjugation efficiency. Antibodies labeled with this tag exhibited detection performance comparable to that of commercial tags, along with excellent long-term storage stability. This study provided a novel metal tag platform featuring controllable structure, simplified preparation process, and stable performance for mass cytometry, which held significant application potential.