Hydraplexing: Design Principles for Fragment-Level Signal Amplification Using a Multi-Amine Model Analyte

Fragment-ion signals are often too weak to support confident quantification at trace levels. This challenge is particularly acute for limited-input measurements, including single cells. We introduce hydraplexing, a fragment-readout strategy that installs multiple isobaric barcodes on a single analyte so that barcode-derived fragment ions add in tandem mass spectrometry (MS2). Unlike conventional isobaric labeling that increases sample plexity, hydraplexing multiplexes barcode-release pathways within a single precursor to amplify signal quantitation. Here, hydraplexing is established using a standalone multiamine model analyte, 1,2,4,5-tetraaminobenzene (TAB), bearing 2-4 tandem mass tags as TAB-TMT2, TAB-TMT3, and TAB-TMT4 derivatives, which isolates barcode multiplicity without coupling the scaffold to target analytes. Using this controlled system, we mapped barcode-release dissociation networks and defined collision-energy regimes that maximize reporter-ion (RI) output while maintaining precursor transmission during higher-energy collisional dissociation. RI output increased with tag number across direct-infusion electrospray as well as liquid chromatography and capillary electrophoresis separations using parallel-reaction monitoring. Complementary fragment ions (TMTc) peaked at lower collision energies and were more condition-sensitive, identifying RIs as the more robust quantitative channel under the conditions tested. This amplification persisted in a peptide-rich single-cell Xenopus laevis digest matrix, where TAB-TMT4 produced an ∼3.5-fold higher RI/precursor response than TAB-TMT2. Hydraplexing was further transferred to 3,4,5-triaminobenzoic acid (TABA), a carboxylate-functionalized scaffold forming 1×-3× TMT derivatives, for which the 2× and 3× derivatives yielded 1.4-fold and 2.7-fold higher RI output than the 1× derivative. Hydraplexing establishes fragment-level signal summation as a tunable design principle for engineering additive tag-derived fragment-ion outputs.