Expanding TMTpro reagents to 32-plex for high-throughput quantitative proteomics on Orbitrap platforms
Dustin Frost, M.D.

 

Tandem Mass Tag™ (TMT™) reagents enable simultaneous identification and relative quantification of thousands of proteins from many samples in a single LC-MS/MS acquisition on Thermo Scientific™ Orbitrap™ platforms. This seminar will highlight the benefits of multiplexing for high-throughput quantitative proteomics applications and introduce the latest advancement in multiplex isobaric tagging technology, the Thermo Scientific™ TMTpro 32plex reagent set. To nearly double the multiplexing of the TMTpro reagent, we developed an additional set of 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 set of tags. We determined the Orbitrap resolving power required to distinguish reporter ions in MS/MS spectra, benchmarked performance for bulk proteome analysis at higher throughput with fewer missing values, characterized chromatographic behavior of the deuterated variants, and established approaches to maximize quantitative performance using the expanded reagent set.

 

 

Expanding Quantitative Proteome Profiling with TMTpro 32-plex Sample Multiplexing
Joao A. Paulo, PhD, MMS

 

Mass spectrometry-based sample multiplexing with isobaric tags permits the development of high-throughput and precise quantitative biological assays with proteome-wide coverage and minimal missing values. Here, we nearly doubled the multiplexing capability of the TMTpro reagent set to up to 35-plex through the incorporation of a single deuterium isotope into the reporter group. We acknowledge that substituting deuterium frequently results in suboptimal peak co-elution which can compromise the accuracy of reporter ion-based quantification. To counteract the deuterium effect on quantitation, we implemented a strategy that necessitated the segregation of non-deuterium and deuterium-containing channels into distinct sub-plexes during normalization procedures with reassembly through a common bridge channel. We highlight several applications of the expanded TMTpro reagent set throughout its development.