Skip to main content

Target Engagement of Small Molecules: Thermal Profiling Approaches on Different Levels

  • Protocol
  • First Online:
Systems Chemical Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1888))


The identification of protein targets and the elucidation of the molecular mechanism of action (MMoA) of bioactive small molecules are central goals of chemical biology. Many different techniques for target identification and engagement are developed, but none of them is generic. Here we describe one of these techniques—the cellular thermal shift assay (CETSA). The assay works without any labeling of proteins or small molecules, which allows the investigation of the unaltered interaction between the interaction partners. Briefly, the influence of small molecules on the thermal stability of proteins within whole cell lysates is investigated. We describe this approach in two variants: the conventional immunoblot-based approach (CETSA), as well as an unbiased approach based on a proteome-wide mass spectrometric analysis (thermal proteome profiling, TPP). The CETSA approach requires preknowledge about possible target proteins and can only detect a few proteins at once. Although TPP is technically more demanding, it allows for the identification of multiple (off)targets without any preknowledge.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others


  1. Ziegler S, Pries V, Hedberg C, Waldmann H (2013) Target identification for small bioactive molecules: finding the needle in the haystack. Angew Chem Int Ed Engl 52(10):2744–2792.

    Article  CAS  PubMed  Google Scholar 

  2. Schurmann M, Janning P, Ziegler S, Waldmann H (2016) Small-molecule target engagement in cells. Cell Chem Biol 23(4):435–441.

    Article  CAS  PubMed  Google Scholar 

  3. Kapoor S, Waldmann H, Ziegler S (2016) Novel approaches to map small molecule-target interactions. Bioorg Med Chem 24(15):3232–3245.

    Article  CAS  PubMed  Google Scholar 

  4. Schenone M, Dancik V, Wagner BK, Clemons PA (2013) Target identification and mechanism of action in chemical biology and drug discovery. Nat Chem Biol 9(4):232–240.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lee H, Lee JW (2016) Target identification for biologically active small molecules using chemical biology approaches. Arch Pharm Res 39(9):1193–1201.

    Article  CAS  PubMed  Google Scholar 

  6. Kanoh N, Honda K, Simizu S, Muroi M, Osada H (2005) Photo-cross-linked small-molecule affinity matrix for facilitating forward and reverse chemical genetics. Angew Chem Int Ed Engl 44(23):3559–3562.

    Article  PubMed  Google Scholar 

  7. Lomenick B, Hao R, Jonai N, Chin RM, Aghajan M, Warburton S, Wang JN, Wu RP, Gomez F, Loo JA, Wohlschlegel JA, Vondriska TM, Pelletier J, Herschman HR, Clardy J, Clarke CF, Huang J (2009) Target identification using drug affinity responsive target stability (DARTS). Proc Natl Acad Sci U S A 106(51):21984–21989.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Savitski MM, Reinhard FB, Franken H, Werner T, Savitski MF, Eberhard D, Martinez Molina D, Jafari R, Dovega RB, Klaeger S, Kuster B, Nordlund P, Bantscheff M, Drewes G (2014) Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 346(6205):1255784.

    Article  CAS  PubMed  Google Scholar 

  9. Martinez MD, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, Sreekumar L, Cao Y, Nordlund P (2013) Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341(6141):84–87. [doi] 341/6141/84 [pii]

    Article  CAS  Google Scholar 

  10. Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ Jr, Wasney GA, Yeung R, Arrowsmith C, Ball LJ, Berglund H, Hui R, Marsden BD, Nordlund P, Sundstrom M, Weigelt J, Edwards AM (2006) Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci U S A 103(43):15835–15840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Franken H, Mathieson T, Childs D, Sweetman GMA, Werner T, Togel I, Doce C, Gade S, Bantscheff M, Drewes G, Reinhard FBM, Huber W, Savitski MM (2015) Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry. Nat Protoc 10(10):1567–1593.

    Article  CAS  PubMed  Google Scholar 

  12. Reinhard FB, Eberhard D, Werner T, Franken H, Childs D, Doce C, Savitski MF, Huber W, Bantscheff M, Savitski MM, Drewes G (2015) Thermal proteome profiling monitors ligand interactions with cellular membrane proteins. Nat Methods 12(12):1129–1131.

    Article  CAS  PubMed  Google Scholar 

  13. Huber KV, Olek KM, Muller AC, Tan CS, Bennett KL, Colinge J, Superti-Furga G (2015) Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling. Nat Methods 12(11):1055–1057.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372.

    Article  CAS  PubMed  Google Scholar 

  15. Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundback T, Nordlund P, Martinez Molina D (2014) The cellular thermal shift assay for evaluating drug target interactions in cells. Nat Protoc 9(9):2100–2122.

    Article  CAS  PubMed  Google Scholar 

  16. Mateus A, Maatta TA, Savitski MM (2016) Thermal proteome profiling: unbiased assessment of protein state through heat-induced stability changes. Proteome Sci 15:13.

    Article  CAS  PubMed  Google Scholar 

  17. Instructions–TMT10plex mass tag labeling kits and reagents. Pierce biotechnology.

Download references


We thank Dr. Marc Schürmann for the establishment of the TPP assay in our department. Without his large effort, it would not have been possible to perform the described assay in-house.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Petra Janning .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Reckzeh, E.S., Brockmeyer, A., Metz, M., Waldmann, H., Janning, P. (2019). Target Engagement of Small Molecules: Thermal Profiling Approaches on Different Levels. In: Ziegler, S., Waldmann, H. (eds) Systems Chemical Biology. Methods in Molecular Biology, vol 1888. Humana Press, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8890-7

  • Online ISBN: 978-1-4939-8891-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics