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

An Orbitrap Eclipse Tribrid mass spectrometer with real time search enhances multiplexed proteome coverage and quantitation accuracy. (#455)

Aaron M Robitaille 1 , Romain Huguet 1 , Ryan Bomgarden 2 , Derek Bailey 1 , Graeme McAlister 1 , Arne Kreutzmann 3 , Daniel Mourad 3 , Woong Kim 1 , John C Rogers 2 , Daniel Lopez-Ferrer 1 , Andreas Huhmer 1 , Vlad Zabrouskov 1 , Zoon Chan 4
  1. Thermo Fisher Scientific, San Jose, CALIFORNIA, United States
  2. Thermo Fisher Scientific, Rockford, IL, USA
  3. Thermo Fisher Scientific, Bremen, Germany
  4. Thermo Fisher Scientific ANZ, SCORESBY, Victoria, Australia

Quantitative proteomics strategies using Tandem Mass Tags™ (TMT™) enable precise measurement of peptides or proteins from samples multiplexed into a single high-resolution LC/MS experiment. Interference can suppress ratio quantitation and thereby mask true differences in abundance. Here we evaluate if an Orbitrap Eclipse Tribrid mass spectrometer including real time search (RTS), advanced spectral processing algorithms, and modified hardware can enhance TMT quantification accuracy and proteome coverage. Synchronous precursor selection (SPS) based methods provided higher accuracy compared to MS2 methods for TMT quantitation. However, depending on which fragments are selected for MS3 quantitation, accuracy can still be distorted. To improve upon this, we implemented RTS between MS2 and MS3 scans. Using this approach, MS3 scans are only triggered if a peptide is identified from the preceding MS2. This increased the number of peptides identified with RTS by 30%. Secondly, RTS selects fragment ions for MS3 quantitation that are generated from the identified peptide on the fly. Thus, quantitation can be improved to be 95% interference free. The Orbitrap Eclipse Tribrid mass spectrometer has an optimized quadrupole that improves ion transmission, enabling narrower isolation widths to improve TMT quantitation accuracy. Additionally, we evaluated how next generation isobaric mass tags could increase multiplexing capacity on the new instrumentation. Overall, the Orbitrap Eclipse™ Tribrid mass spectrometer includes features such as TurboTMT and Precursor Fit which facilitate intelligent acquisition methods that improve TMT quantitation accuracy, precision, and proteome coverage.