Multiplexed quantitative proteomics has gained significant attention in various research domains, including chemoproteomics, drug discovery, single-cell analysis, and plasma profiling. To address the demand for higher multiplexing capabilities and improved sample throughput, a novel TMTpro 32-plex reagent was developed and recently introduced, allowing for extensive protein quantification with enhanced accuracy and precision.

To fully leverage the potential of the TMTpro 32plex reagent, it is crucial to employ mass spectrometers capable of resolving 3 mDa spaced reporter ion peaks. Consequently, the Thermo Scientific Orbitrap Ascend MultiOmics Tribrid mass spectrometer now offers new extended resolving power (RP) settings at Turbo 45k, Turbo 60k, eFT 75k, and eFT 90k, ensuring optimal resolution and speed for accurate quantification of proteins and peptides in highly multiplexed experiments.

One of the key features that enhances data acquisition efficiency while maintaining quantification accuracy is the Real-Time Search (RTS) feature, implemented and available on Orbitrap Tribrid MS systems. RTS enables on-the-fly peptide spectrum matches (PSMs) for MS2 spectra with online database search, triggering Synchronous Precursor Selection (SPS)-MS3 only upon confident identifications. This feature significantly enhances data acquisition and quantification efficiency in complex sample mixtures.

Our experiments utilized the Orbitrap Ascend MultiOmics Tribrid MS and Vanquish Neo systems with a PepMap 50cm column, employing a 140-minute LC gradient length with about 1ug sample loaded on column. Both MS2 and Real-Time Search MS3 experiments were performed under Turbo 45k, Turbo 60k, eFT 75k, and eFT 90k resolving power settings, with optimized maximum injection time for each resolving power and an AGC target of 300%. Data analysis was performed using Proteome Discoverer 3.2 TMTpro 32 plex quan node.

The sample set comprised TMTpro 32plex-labeled HeLa digest with 1:1 and 1:4 ratios across channels, enabling assessment of the instrument parameters necessary to resolve reporter ions. Additionally, TMTpro-labeled HeLa digest with a Pierce 6 protein digest mix was used, with HeLa digest labeled 1:1 across all channels and the protein mix spiked in at different ratios across channels.

Preliminary results from our TMTpro 32-plex experiment revealed quantification of over 4000 protein groups and more than 22,000 quantified peptides in the MS2 experiments. Evaluation with RTS-SPS-MS3 further demonstrated improved quantification accuracy. Instrument parameters, including resolving power, AGC, and injection time, were optimized. Comparison between 16-plex and 32-plex label experiments indicated equal or greater quantified proteins/peptides in the 32-plex experiment compared to two sets of 16-plex experiments. Quantification using ratio reference within sets exhibited accurate and precise results.