Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Going Beyond 11: TMTpro 16plex Regents for Higher Quantitative Proteomic Sample Multiplexing (#192)

Ryan Bomgarden 1 , Sergei Snovida 1 , Amarjeet Flora 1 , Leigh Foster 1 , Rosa Viner 2 , Aaron Robitaille 2 , Andrew Thompson Andrew Thompson 3 , Karsten Kuhn 3 , Ian Pike 3 , John Rogers 1
  1. Thermo Fisher Scientific, Rockford, IL, United States
  2. Thermo Fisher Scientific, San Jose, CA, United States
  3. Proteome Sciences, London, WC1H 9BB, UK


Thermo Scientific Tandem Mass Tag® (TMT®) Reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry. The maximum number of samples that can be compared in a single experiment is current limited to eleven by the number of stable isotopes incorporated into the reporter and mass balance region. Here, we describe a new set of mass tags (TMTpro regents) with a proline-based reporter and longer linker which can support higher sample multiplexing for up to sixteen samples.


Cellular protein digests were labeled with TMT 11plex or TMTpro 16plex reagents using standard methods. Samples were analyzed by LC-MS to assess reporter ion fragmentation, labeling efficiency and peptide/protein identification rates. To assess protein quantitative accuracy and precision, HeLa protein digest was spiked with Pierce 6 protein mix at distinct ratios and labeled with different tags. Samples were analyzed on Orbitrap hybrid and tribrid instruments using MS2 or SPS-MS3 methods. Data analysis was performed using Thermo Scientific Proteome Discoverer™ 2.4.

Results and Discussion

Increasing the number of samples that can be compared in a single LC-MS experiment is highly desired to improve sample throughput, assess multiple experimental conditions and decrease missing values among replicates. We have designed a new tandem mass tag set that can be used to concurrently quantify up to sixteen protein samples. The new tag contains nine stable isotopes and a novel proline-based reporter with the same elemental composition and mass as the TMT reporter ion. Compared to TMT reagents, TMTpro reagents have the same peptide labeling efficiency, but require slightly lower collision energy for reporter ion fragmentation. We also demonstrate that TMTpro 16plex samples have similar peptide/protein identification rates compared to TMT 11plex samples, exhibit a 20-fold dynamic range of quantitation and are compatible with standard enrichment and fractionation workflows.