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

Real-time, high density monitoring of pTyr signaling targets in human tumors using SureQuant heavy peptide triggered targeted quantitation (#861)

Lauren E Stopfer 1 2 , Cameron T Flower 1 2 , Aaron S Gajadhar 3 , Bhavin Patel 4 , Sebastien Gallien 5 , Romain Huguet 3 , Graeme McAlister 3 , Derek Bailey 3 , Shannon Eliuk 3 , Markus Kellmann 6 , Tabiwang N Arrey 6 , Alexander Harder 6 , Daniel Lopez-Ferrer 3 , Andreas Huhmer 3 , Forest M White 1 2
  1. Massachusetts Institute of Technology, Cambridge, MA, United States
  2. Koch Institute for Integrative Cancer Research, Cambridge, MA, United States
  3. Thermo Fisher Scientific, San Jose, CA, United States
  4. Thermo Fisher Scientific, Rockford, IL, United States
  5. Precision Medicine Science Center, Thermo Fisher Scientific, Cambridge, MA, United States
  6. Thermo Fisher Scientific, Bremen, Germany

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer.  Profiling the tumor pTyr proteome may reveal therapeutic insights critical to combating disease. Existing discovery-based mass spectrometry methods to monitor pTyr networks favor broad coverage while sacrificing reproducibility, while targeted methods attempt to address reproducibility but are focused on a limited subset of sites in the entire pTyr network. To achieve high coverage, high reproducibility analysis of the network, we applied a novel, high-density global targeted approach that leverages isotopically-labeled trigger peptides to reliably quantify several hundred commonly dysregulated pTyr targets in a cohort of 30 human colorectal tumor samples.

Tumor specimens were lysed, and proteins were reduced, alkylated, and digested to peptides. A mixture of stable isotopically-labeled (SIL) peptides, corresponding to several hundred pTyr targets, was spiked into each tryptic tumor digest. The endogenous and SIL forms of pTyr-containing peptides were isolated with a 2-step immunoprecipitation and IMAC enrichment, and LC-MS analysis was performed with IonOpticks Aurora column (250mm x 0.075mm) and an EASY-nLC™ 1200 coupled to an Orbitrap Exploris™ 480 mass spectrometer. To ensure reproducible measurement of selected pTyr targets, a SureQuantTMmethod adapted from the internal standard triggered parallel reaction monitoring method (IS-PRM) was performed where real-time heavy peptide detection triggered selective and sensitive measurements of endogenous pTyr targets. Data analysis was performed using Proteome Discoverer and Skyline software.

SIL-triggered targeted pTyr analysis quantified over 300 unique pTyr across patient tumor samples, revealing quantitatively distinct proteomic signatures. In some cases, pTyr profiles align with proteomic & transcriptomic molecular subtypes previously reported. However, pTyr profiling also revealed putative patient specific oncogenic driving mechanisms not captured in global proteomics. Heavy peptide triggered and guided acquisition maximizes the efficiency of targeted quantification by enhancing the detectability of targets, significantly improving measurement reproducibility across analysis.