Cellular functions arise from the coordinated action of proteoforms, which typically form multiproteoform complexes (MPCs), rather than functioning as isolated molecular entities. Deciphering the architecture and composition of MPCs is essential for linking proteoform diversity to biological function. Native top-down (nTD MS) and complex-down mass spectrometry (CxD MS) have emerged as powerful strategies to characterize MPCs, offering intact mass analysis as well as gas-phase sequencing either at the level of the complete assembly or its constituent proteoform subunits. Because the attainable sequence coverage is highly influenced by the ion activation technique, expanding activation strategies is key to improving proteoform characterization. To this end, we implemented infrared (IR) activation for the analysis of soluble MPCs─alcohol dehydrogenase (ADH; 147 kDa tetramer), enolase (96 kDa dimer), and pyruvate kinase (PK; 232 kDa tetramer). IR photons were used to induce infrared multiphoton dissociation (IRMPD) and to enhance electron-based fragmentation via activated-ion electron transfer dissociation (AI-ETD), and performance was benchmarked against higher-energy collisional dissociation (HCD). For ADH (∼36 kDa subunits), AI-ETD, HCD, and IRMPD returned similar sequence coverages in nTD MS experiments (36, 38, and 34%, respectively), with complementary cleavages resulting in a combined 48% coverage. As subunit mass increased, radical-driven fragmentation provided a clear advantage: for PK (∼57 kDa subunits), AI-ETD achieved 28% sequence coverage─approximately 15% higher than HCD or IRMPD. Together, these results highlight IR irradiation─both as a standalone dissociation modality and as a complement to electron-based activation─as a versatile strategy to enhance proteoform-level sequencing in native and complex-down MS workflows.
Nagy et al. (Tue,) studied this question.
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