Utrecht U Targeted Proteomics - Veth_et_al_2023

Elucidating Fibroblast Growth Factor-induced kinome dynamics using targeted mass spectrometry and dynamic modeling
Data License: CC BY 4.0 | ProteomeXchange: PXD039005 | doi: https://doi.org/10.6069/b4ab-er53
  • Organism: Homo sapiens
  • Instrument: TSQ Altis
  • SpikeIn: Yes
  • Keywords: Phosphoproteomics; kinase; targeted mass spectrometry; kinase dynamics; fibroblast growth factors; dynamic modeling; fibroblast growth factor receptor; breast cancer. 
  • Lab head: Maarten Altelaar Submitter: Tim Veth
Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that when activated by their ligands, can elicit a wide range of healthy and disease-related processes such as cell proliferation and the epithelial-to-mesenchymal transition (EMT). The detailed molecular pathway dynamics that coordinate these responses have remained unclear. To elucidate and monitor these dynamics, we stimulated MCF-7 breast cancer cells with either FGF2, 3, 4, 10, or 19. Following activation of the receptor, we quantified the kinase activity dynamics of 44 kinases using a targeted mass spectrometry assay. Our system-wide kinase activity data, supplemented with (phopsho)proteomics data, reveal ligand-dependent distinct pathway dynamics, elucidate the involvement of not earlier reported kinases such as MARK, and revise some of the pathway effects on biological outcomes. In addition, logic-based dynamic modeling of the kinome dynamics further verifies the biological goodness-of-fit of the predicted models and reveals tight regulation of the RAF kinase family.
Experiment Description
FGF-stimulated MCF-7 cells were digested using trypsin and LysC, heavy labeled phosphopeptides were added, and the samples were desalted and enriched for phosphopeptides. Subsequently, the samples were measured on a Thermo Altis.
Created on 12/20/22, 10:25 PM
Clustergrammer Heatmap
PRM_HL_library_last_batch_2022-12-19_11-30-24.sky.zip2022-12-20 22:25:002789998908
2nd_batch_peptides_PRM_2022-12-19_11-29-04.sky.zip2022-12-20 22:25:001330303102
FGFR_method_JPT1_HLlib_2022-12-19_11-27-59.sky.zip2022-12-20 22:25:001101761818,6895
FGFR_A-loop-iRT_FGF10_FGF19_clean_2022-12-14_10-44-48.sky.zip2022-12-20 22:25:0030447829830
FGFR_A-loop-iRT_FGF3_FGF4_clean_2022-12-14_10-43-52.sky.zip2022-12-20 22:25:0030498834630
FGFR_A-loop-iRT_found_2022-12-14_10-42-47.sky.zip2022-12-20 22:25:00426111245215

For the following libraries a parallel reaction monitoring (PRM) approach was used to measure the synthetic peptides. Subsequently, the raw files were searched in various MaxQuant runs, separated based on the heavy labeled amino acid modification to keep the search space within reasonable limits. The search results were loaded into Skyline to visualize and manually validate the identified peaks. Then, synthetic MS2 spectra were exported from Skyline and used as a library in the SRM experiments.

  • 2nd_batch_peptides_PRM_PseudoMS2.blib
  • FGFR_method_JPT1_HLlib_PseudoMS2.blib
  • PRM_HL_library_last_batch_PseudoMS2.blib


Source files used to build the following libraries are available at https://db.systemsbiology.net/sbeams/cgi/PeptideAtlas/PASS_View?identifier=PASS01234. Spectral library generation is described in the associated publication: High-Throughput Assessment of Kinome-wide Activation States.

  • 02A11_AL_jpt_DDA_TripleTOF2.blib
  • 02A11_AL_jpt_DDA_TripleTOF3.blib
  • HpH_library_phosphoRS.blib
  • TSQ_DDA_AL.blib