Related Experiment Video
Updated: Aug 5, 2026
Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)
Published on: December 18, 2020
Implementation of Nonisobaric TMT Analogs for Accurate Precursor-Level Quantification by plexDIA
1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
Rationale:
Multiplexed 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.
Methods:
Three 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.
Results:
Over 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+).
Conclusions:
TMTpro 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.
