Implementation of Nonisobaric TMT Analogs for Accurate Precursor-Level Quantification by plexDIA

Area of Science:

• Proteomics
• Mass Spectrometry
• Biotechnology

Background:

• Multiplexed quantitative proteomics increases sample throughput and reduces resource needs.
• Integrating sample multiplexing with data-independent acquisition (DIA) presents significant challenges.
• A novel strategy, TMTpro plex-DIA, is introduced to address these integration challenges.

Purpose of the Study:

• To present and validate the TMTpro plex-DIA strategy for multiplexed quantitative proteomics.
• To leverage MS1-level mass differences of TMTpro reagents for quantification.
• To enable high-throughput proteomics without compromising DIA sensitivity.

Main Methods:

• Three Saccharomyces cerevisiae deletion strains were labeled with TMTpro reagents (light, standard, heavy).
• Samples were mixed in permutations and analyzed using narrow-window DIA on an Orbitrap Astral mass spectrometer.
• Data analysis was performed using FragPipe/MSFragger with plex-DIA quantification.

Main Results:

• Over 2000 protein groups and 20,000 precursors were identified per channel.
• Identification rates were consistent across all three TMTpro channels.
• All nine expected deletion patterns were accurately identified, confirming channel-specific depletions.

Conclusions:

• TMTpro plex-DIA enables accurate, multiplexed quantitative proteomics via MS1-level mass separation.
• Characteristic deletion patterns act as molecular barcodes, validating sample identity.
• The plex-DIA approach is broadly applicable for high-throughput, multiplexed proteomics.