Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Simultaneous targeted and discovery phosphoproteomics of cell signaling pathways using novel hybrid-DIA acquisition strategy (#187)

Ana Martínez-Val 1 , Kyle Fort 2 , Andreas F. Huhmer 3 , Yue Xuan 2 , Alexander Makarov 2 , Jesper V. Olsen 1 , Enzo Huang 4
  1. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DENMARK
  2. Thermo Fisher Scientific, Bremen, Germany
  3. Thermo Fisher Scientific, San Jose, CA, USA
  4. Thermo Fisher Scientific, Scoresby, VIC, Australia

Due to low stoichiometry of regulatory phosphorylation sites and high complexity of the phosphoproteome, achieving sufficient coverage by MS to cover all phosphorylation sites of interest is challenging. Targeted methods such SureQuantTM has proven useful in this context, allowing the precise quantification of dozens of targets even with limited sample amount. Here, we present a Hybrid-DIA method to extract precise quantitative information of selected phosphopeptide targets in parallel with profiling the global phosphoproteome in just one MS run per sample.

SureQuant™ Multipathway Phosphopeptide Standard (Thermo) combines 131 heavy-labeled tryptic phosphopeptides that correspond to phosphorylation sites of six relevant signaling pathways (EGFR, RAS-MAPK, P3IK/AKT/mTOR, AMPK, apoptosis and stress). Previously, using that mixture we were able to monitor phosphorylation changes in response to EGF and IGF-alpha in SY5Y cells. Here, we explored the sensitivity and detection limits of SureQuant™ method by using decreasing amounts (50, 25, 12 and 6 µg) of tryptic HeLa peptides for phospho-enrichment. Taking advantage of the higher sensitivity afforded by low-flow rate WHISPER gradients, we found that with 6 µg, SureQuant™ method provides quantitative information for 80 out of the 131 phosphorylation sites targeted.

Next, we benchmarked Hybrid-DIA against SureQuant™ to assess the potential to extract information from the full phosphoproteome in parallel with targeting the >130 phosphopeptides on the inclusion list. HeLa cells were grown in p6 dishes and treated 15 minutes with three kinase inhibitors: EGFRi (Lapatinib), MEKi (PD0325901) and P3Ki (Wortmannin), followed by stimulation with 100ng/ml of EGF for 10 and 90 minutes. The recovered peptides were mixed with 0.05 pmol of the Multipathway Phosphopeptide Standard mix and phosphopeptide-enriched with TiIMACHP beads. Samples were analyzed using either SureQuant™ or Hybrid-DIA using a 20 samples/day WHISPER gradient. Hybrid-DIA results recapitulated those obtained using SureQuant™ in terms of accuracy and sensitivity for the selected targets, whilst also providing information of ~4,000 phosphorylation sites from the global phosphoproteome. Importantly, the unbiased DIA analysis allowed us to identify ~200 phosphorylation sites differentially regulated by EGF and/or kinase inhibitors, revealing clusters of novel sites with specific kinase trends.

These results reveal the potential of the combination of targeted and discovery proteomics to maximize the information derived from phosphoproteomics experiments, which can be highly relevant in experimental contexts where samples with limited amount do not allow for multiple MS runs.