IDEAS home Printed from https://ideas.repec.org/a/nat/natcom/v16y2025i1d10.1038_s41467-025-59404-y.html
   My bibliography  Save this article

Spike-in enhanced phosphoproteomics uncovers synergistic signaling responses to MEK inhibition in colon cancer cells

Author

Listed:
  • Mirjam Bentum

    (Max Delbrück Center for Molecular Medicine
    Humboldt-Universität zu Berlin)

  • Bertram Klinger

    (Humboldt-Universität zu Berlin
    Charité-Universitätsmedizin Berlin)

  • Anja Sieber

    (Humboldt-Universität zu Berlin
    Charité-Universitätsmedizin Berlin)

  • Sheyda Naghiloo

    (Max Delbrück Center for Molecular Medicine)

  • Henrik Zauber

    (Max Delbrück Center for Molecular Medicine)

  • Nadine Lehmann

    (Charité-Universitätsmedizin Berlin)

  • Mohamed Haji

    (Max Delbrück Center for Molecular Medicine)

  • Sylvia Niquet

    (Max Delbrück Center for Molecular Medicine
    Berlin Institute of Health)

  • Philipp Mertins

    (Max Delbrück Center for Molecular Medicine
    Berlin Institute of Health)

  • Nils Blüthgen

    (Humboldt-Universität zu Berlin
    Charité-Universitätsmedizin Berlin)

  • Matthias Selbach

    (Max Delbrück Center for Molecular Medicine
    Charité-Universitätsmedizin Berlin)

Abstract

Targeted kinase inhibitors are a cornerstone of cancer therapy, but their success is often hindered by the complexity of cellular signaling networks that can lead to resistance. Overcoming this challenge necessitates a deep understanding of cellular signaling responses. While standard global phosphoproteomics offers extensive insights, lengthy processing times, the complexity of data interpretation, and frequent omission of crucial phosphorylation sites limit its utility. Here, we combine data-independent acquisition (DIA) with spike-in of synthetic heavy stable isotope-labeled phosphopeptides to facilitate the targeted detection of particularly informative phosphorylation sites. Our spike-in enhanced detection in DIA (SPIED-DIA) approach integrates the improved sensitivity of spike-in-based targeted detection with the discovery potential of global phosphoproteomics into a simple workflow. We employed this method to investigate synergistic signaling responses in colorectal cancer cell lines following MEK inhibition. Our findings highlight that combining MEK inhibition with growth factor stimulation synergistically activates JNK signaling in HCT116 cells. This synergy emphasizes the therapeutic potential of concurrently targeting MEK and JNK pathways, as evidenced by the significantly impaired growth of HCT116 cells when treated with both inhibitors. Our results demonstrate that SPIED-DIA effectively identifies synergistic signaling responses in colorectal cancer cells, presenting a valuable tool for uncovering new therapeutic targets and strategies in cancer treatment.

Suggested Citation

  • Mirjam Bentum & Bertram Klinger & Anja Sieber & Sheyda Naghiloo & Henrik Zauber & Nadine Lehmann & Mohamed Haji & Sylvia Niquet & Philipp Mertins & Nils Blüthgen & Matthias Selbach, 2025. "Spike-in enhanced phosphoproteomics uncovers synergistic signaling responses to MEK inhibition in colon cancer cells," Nature Communications, Nature, vol. 16(1), pages 1-16, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-59404-y
    DOI: 10.1038/s41467-025-59404-y
    as

    Download full text from publisher

    File URL: https://www.nature.com/articles/s41467-025-59404-y
    File Function: Abstract
    Download Restriction: no

    File URL: https://libkey.io/10.1038/s41467-025-59404-y?utm_source=ideas
    LibKey link: if access is restricted and if your library uses this service, LibKey will redirect you to where you can use your library subscription to access this item
    ---><---

    References listed on IDEAS

    as
    1. Mathurin Dorel & Bertram Klinger & Tommaso Mari & Joern Toedling & Eric Blanc & Clemens Messerschmidt & Michal Nadler-Holly & Matthias Ziehm & Anja Sieber & Falk Hertwig & Dieter Beule & Angelika Egge, 2021. "Neuroblastoma signalling models unveil combination therapies targeting feedback-mediated resistance," PLOS Computational Biology, Public Library of Science, vol. 17(11), pages 1-26, November.
    2. Martin Frejno & Chen Meng & Benjamin Ruprecht & Thomas Oellerich & Sebastian Scheich & Karin Kleigrewe & Enken Drecoll & Patroklos Samaras & Alexander Hogrebe & Dominic Helm & Julia Mergner & Jana Zec, 2020. "Proteome activity landscapes of tumor cell lines determine drug responses," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
    3. Corinna Friedrich & Simon Schallenberg & Marieluise Kirchner & Matthias Ziehm & Sylvia Niquet & Mohamed Haji & Christin Beier & Jens Neudecker & Frederick Klauschen & Philipp Mertins, 2021. "Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    4. Ruedi Aebersold & Matthias Mann, 2016. "Mass-spectrometric exploration of proteome structure and function," Nature, Nature, vol. 537(7620), pages 347-355, September.
    5. Philipp Mertins & D. R. Mani & Kelly V. Ruggles & Michael A. Gillette & Karl R. Clauser & Pei Wang & Xianlong Wang & Jana W. Qiao & Song Cao & Francesca Petralia & Emily Kawaler & Filip Mundt & Karste, 2016. "Proteogenomics connects somatic mutations to signalling in breast cancer," Nature, Nature, vol. 534(7605), pages 55-62, June.
    6. Anirudh Prahallad & Chong Sun & Sidong Huang & Federica Di Nicolantonio & Ramon Salazar & Davide Zecchin & Roderick L. Beijersbergen & Alberto Bardelli & René Bernards, 2012. "Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR," Nature, Nature, vol. 483(7387), pages 100-103, March.
    7. Vadim Demichev & Lukasz Szyrwiel & Fengchao Yu & Guo Ci Teo & George Rosenberger & Agathe Niewienda & Daniela Ludwig & Jens Decker & Stephanie Kaspar-Schoenefeld & Kathryn S. Lilley & Michael Mülleder, 2022. "dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts," Nature Communications, Nature, vol. 13(1), pages 1-8, December.
    Full references (including those not matched with items on IDEAS)

    Most related items

    These are the items that most often cite the same works as this one and are cited by the same works as this one.
    1. Kai Li & Guo Ci Teo & Kevin L. Yang & Fengchao Yu & Alexey I. Nesvizhskii, 2025. "diaTracer enables spectrum-centric analysis of diaPASEF proteomics data," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    2. Huanhuan Gao & Yi Zhu & Dongxue Wang & Zongxiang Nie & He Wang & Guibin Wang & Shuang Liang & Yuting Xie & Yingying Sun & Wenhao Jiang & Zhen Dong & Liqin Qian & Xufei Wang & Mengdi Liang & Min Chen &, 2025. "iDIA-QC: AI-empowered data-independent acquisition mass spectrometry-based quality control," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    3. Yi Yang & Qun Fang, 2024. "Prediction of glycopeptide fragment mass spectra by deep learning," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    4. Fengchao Yu & Guo Ci Teo & Andy T. Kong & Klemens Fröhlich & Ginny Xiaohe Li & Vadim Demichev & Alexey I. Nesvizhskii, 2023. "Analysis of DIA proteomics data using MSFragger-DIA and FragPipe computational platform," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    5. Mathurin Dorel & Bertram Klinger & Tommaso Mari & Joern Toedling & Eric Blanc & Clemens Messerschmidt & Michal Nadler-Holly & Matthias Ziehm & Anja Sieber & Falk Hertwig & Dieter Beule & Angelika Egge, 2021. "Neuroblastoma signalling models unveil combination therapies targeting feedback-mediated resistance," PLOS Computational Biology, Public Library of Science, vol. 17(11), pages 1-26, November.
    6. Katrin Stuber & Tobias Schneider & Jill Werner & Michael Kovermann & Andreas Marx & Martin Scheffner, 2021. "Structural and functional consequences of NEDD8 phosphorylation," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    7. Gholamreza Safaee Ardekani & Seyed Mehdi Jafarnejad & Larry Tan & Ardavan Saeedi & Gang Li, 2012. "The Prognostic Value of BRAF Mutation in Colorectal Cancer and Melanoma: A Systematic Review and Meta-Analysis," PLOS ONE, Public Library of Science, vol. 7(10), pages 1-10, October.
    8. Chengxin Dai & Anja Füllgrabe & Julianus Pfeuffer & Elizaveta M. Solovyeva & Jingwen Deng & Pablo Moreno & Selvakumar Kamatchinathan & Deepti Jaiswal Kundu & Nancy George & Silvie Fexova & Björn Grüni, 2021. "A proteomics sample metadata representation for multiomics integration and big data analysis," Nature Communications, Nature, vol. 12(1), pages 1-8, December.
    9. Guilherme Reis-de-Oliveira & Victor Corasolla Carregari & Gabriel Rodrigues dos Reis de Sousa & Daniel Martins-de-Souza, 2024. "OmicScope unravels systems-level insights from quantitative proteomics data," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    10. Valentina Cordo’ & Mariska T. Meijer & Rico Hagelaar & Richard R. Goeij-de Haas & Vera M. Poort & Alex A. Henneman & Sander R. Piersma & Thang V. Pham & Koichi Oshima & Adolfo A. Ferrando & Guido J. R, 2022. "Phosphoproteomic profiling of T cell acute lymphoblastic leukemia reveals targetable kinases and combination treatment strategies," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    11. Reta Birhanu Kitata & Marija Velickovic & Zhangyang Xu & Rui Zhao & David Scholten & Rosalie K. Chu & Daniel J. Orton & William B. Chrisler & Tong Zhang & Jeremy V. Mathews & Benjamin M. Bumgarner & D, 2025. "Robust collection and processing for label-free single voxel proteomics," Nature Communications, Nature, vol. 16(1), pages 1-15, December.
    12. Sofani Tafesse Gebreyesus & Asad Ali Siyal & Reta Birhanu Kitata & Eric Sheng-Wen Chen & Bayarmaa Enkhbayar & Takashi Angata & Kuo-I Lin & Yu-Ju Chen & Hsiung-Lin Tu, 2022. "Streamlined single-cell proteomics by an integrated microfluidic chip and data-independent acquisition mass spectrometry," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    13. A. Contreras-Sanz & G. L. Negri & M. J. Reike & H. Z. Oo & J. M. Scurll & S. E. Spencer & K. Nielsen & K. Ikeda & G. Wang & C. L. Jackson & S. Gupta & M. E. Roberts & D. M. Berman & R. Seiler & G. B. , 2025. "Proteomic profiling identifies muscle-invasive bladder cancers with distinct biology and responses to platinum-based chemotherapy," Nature Communications, Nature, vol. 16(1), pages 1-18, December.
    14. Jonathan J. Swietlik & Stefanie Bärthel & Chiara Falcomatà & Diana Fink & Ankit Sinha & Jingyuan Cheng & Stefan Ebner & Peter Landgraf & Daniela C. Dieterich & Henrik Daub & Dieter Saur & Felix Meissn, 2023. "Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation," Nature Communications, Nature, vol. 14(1), pages 1-17, December.
    15. Melih Yilmaz & William E. Fondrie & Wout Bittremieux & Carlo F. Melendez & Rowan Nelson & Varun Ananth & Sewoong Oh & William Stafford Noble, 2024. "Sequence-to-sequence translation from mass spectra to peptides with a transformer model," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    16. Jennifer G. Abelin & Erik J. Bergstrom & Keith D. Rivera & Hannah B. Taylor & Susan Klaeger & Charles Xu & Eva K. Verzani & C. Jackson White & Hilina B. Woldemichael & Maya Virshup & Meagan E. Olive &, 2023. "Workflow enabling deepscale immunopeptidome, proteome, ubiquitylome, phosphoproteome, and acetylome analyses of sample-limited tissues," Nature Communications, Nature, vol. 14(1), pages 1-22, December.
    17. Yiqun Zhang & Fengju Chen & Darshan S. Chandrashekar & Sooryanarayana Varambally & Chad J. Creighton, 2022. "Proteogenomic characterization of 2002 human cancers reveals pan-cancer molecular subtypes and associated pathways," Nature Communications, Nature, vol. 13(1), pages 1-19, December.
    18. Hao Hu & Wei Hu & An-Di Guo & Linhui Zhai & Song Ma & Hui-Jun Nie & Bin-Shan Zhou & Tianxian Liu & Xinglong Jia & Xing Liu & Xuebiao Yao & Minjia Tan & Xiao-Hua Chen, 2024. "Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    19. Kenny K. H. Yu & Sreyashi Basu & Gerard Baquer & Ryuhjin Ahn & Jennifer Gantchev & Sonali Jindal & Michael S. Regan & Zaki Abou-Mrad & Michael C. Prabhu & Marc J. Williams & Alicia D. D’Souza & Seth W, 2025. "Investigative needle core biopsies support multimodal deep-data generation in glioblastoma," Nature Communications, Nature, vol. 16(1), pages 1-23, December.
    20. Yuwan Chen & Wen Zhou & Yufei Xia & Weijie Zhang & Qun Zhao & Xinwei Li & Hang Gao & Zhen Liang & Guanghui Ma & Kaiguang Yang & Lihua Zhang & Yukui Zhang, 2023. "Targeted cross-linker delivery for the in situ mapping of protein conformations and interactions in mitochondria," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

    More about this item

    Statistics

    Access and download statistics

    Corrections

    All material on this site has been provided by the respective publishers and authors. You can help correct errors and omissions. When requesting a correction, please mention this item's handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-59404-y. See general information about how to correct material in RePEc.

    If you have authored this item and are not yet registered with RePEc, we encourage you to do it here. This allows to link your profile to this item. It also allows you to accept potential citations to this item that we are uncertain about.

    If CitEc recognized a bibliographic reference but did not link an item in RePEc to it, you can help with this form .

    If you know of missing items citing this one, you can help us creating those links by adding the relevant references in the same way as above, for each refering item. If you are a registered author of this item, you may also want to check the "citations" tab in your RePEc Author Service profile, as there may be some citations waiting for confirmation.

    For technical questions regarding this item, or to correct its authors, title, abstract, bibliographic or download information, contact: Sonal Shukla or Springer Nature Abstracting and Indexing (email available below). General contact details of provider: http://www.nature.com .

    Please note that corrections may take a couple of weeks to filter through the various RePEc services.

    IDEAS is a RePEc service. RePEc uses bibliographic data supplied by the respective publishers.