TMTpro reagents are popular reagents for highly multiplexed mass spectrometry (MS)-based proteomics, enabling relative quantification of many samples in a single LC-MS/MS acquisition. 18-plex TMTpro reagents have been previously demonstrated to provide accurate and precise quantification of proteins and peptides by high-resolution LC-MS/MS analysis on Orbitrap platforms. Each tag in the multiplex set incorporates nine 13C & 15N stable isotopes at unique positions within the structure to enable generation of reporter ions during MS/MS fragmentation with distinct masses that differ by a minimum of 6 mDa.

To further increase multiplexing capacity, we designed 17 structurally identical isotopologues that substitute one 2H stable isotope into the reporter group to impart a 3 mDa mass difference between the new variants and the existing 18-plex tags. We strategically located this deuterium isotope on the isobutyl group proximal to the nitrogen atom on the reporter to minimize chromatographic shifts and for synthetic tractability using the existing synthesis route and isotopic starting materials. The deuterated TMTpro reagents were synthesized using existing manufacturing methods and chemical purity & isotopic incorporation were verified by direct infusion MS.

BSA digest and HeLa digest samples labeled with deuterated and non-deuterated TMTpro reagents were subjected to LC-MS/MS analysis using a Vanquish Neo UPLC with a 50cm EASY-Spray HPLC column interfaced with an Orbitrap Eclipse MS system. MS2and RTS SPS-MS3 acquisitions were performed at various eFT and TurboTMT resolving powers. Data was processed with Proteome Discoverer to determine labeling efficiency and numbers of identified & quantified peptides & proteins.

BSA digest labeled with a subset of tags was analyzed using a targeted MS/MS acquisition method to evaluate chromatographic shifts between peptides labeled with the deuterated and non-deuterated tags and to determine the impact on quantitative metrics. HeLa digest samples were labeled with 32-plex TMTpro reagents to determine appropriate instrument parameters, evaluate peptide identification rates, and assess quantitative performance. An Orbitrap resolving power (RP) of ≥90K was sufficient to baseline resolve the reporter ions differing in mass by 3 mDa in MS/MS spectra, while TurboTMT acquisition at RP 45K permitted distinguishing reporter ions with greater instrument duty cycle efficiency to realize greater proteomic depth. Our preliminary LC-MS/MS data using 18-plex and 32-plex TMTpro reagents demonstrate comparable labeling efficiency, peptide identification rates, and quantitative performance with fewer missing values for the expanded reagent set.