ArticleRapid communications in mass spectrometry : RCM2026
Implementation of Nonisobaric TMT Analogs for Accurate Precursor-Level Quantification by plexDIA.
Article in Rapid communications in mass spectrometry : RCM, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
rationaleMultiplexed quantitative proteomics enables simultaneous analysis of multiple biological samples, increasing throughput while reducing instrument time and sample requirements. However, integrating sample multiplexing with data-independent acquisition (DIA) remains challenging. We present a TMTpro plexDIA strategy leveraging MS1-level mass differences between nonisobaric TMTpro reagent variants to enable multiplexed quantification without compromising DIA sensitivity.
methodsThree Saccharomyces cerevisiae deletion strains (Δmet6, Δpfk2, and Δura2) were labeled with TMTproZero (light), TMTpro16 (standard), and super-heavy TMTpro (heavy), mixed in three permutations, and analyzed by narrow-window DIA (2 m/z isolation, 300 scan events) on an Orbitrap Astral mass spectrometer. Database searching was performed using FragPipe/MSFragger with plex-DIA quantification enabled.
resultsOver 2000 protein groups and over 20 000 precursors were identified per channel per mixture, with closely matched identification rates across all three channels. All nine expected deletion patterns were correctly identified, with channel-specific depletion reproduced consistently across precursor charge states (2+, 3+, and 4+).
conclusionsTMTpro plex-DIA enables accurate, multiplexed quantitative proteomics through MS1-level mass separation of the nonisobaric TMTpro isotopologs. The characteristic deletion patterns observed for each knockout strain serve as intrinsic molecular barcodes, validating sample identity and demonstrating the broad utility of plex-DIA for high-throughput, multiplexed proteomics applications.
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