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Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics

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Mass Spectrometry-Based Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2718))

Abstract

Terminal amine isotopic labeling of substrates (TAILS) is a sensitive and robust quantitative mass spectrometry (MS)-based proteomics method used for the characterization of physiological or proteolytically processed protein N-termini, as well as other N-terminal posttranslational modifications (PTMs). TAILS is a well-established, high-throughput, negative enrichment workflow that enables system-wide exploration of N-terminomes independent of sample complexity. TAILS makes use of amine reactivity of free N-termini and a highly efficient aldehyde-functionalized polymer to deplete internal peptides generated after proteolytic digestion during sample preparation. Thereby, it enriches for natural N-termini, allowing for unbiased and complete investigation of differential proteolysis, protease substrate discovery, and analysis of N-terminal PTMs. In this chapter, we provide a state-of-the-art protocol, with detailed steps in all parts of the TAILS sample preparation, MS analysis, and post-processing of acquired data.

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References

  1. Aebersold R, Mann M (2016) Mass-spectrometric exploration of proteome structure and function. Nature 537:347–355. https://doi.org/10.1038/nature19949

    Article  CAS  PubMed  Google Scholar 

  2. Bond JS (2019) Proteases: history, discovery, and roles in health and disease. J Biol Chem 294:1643–1651. https://doi.org/10.1074/jbc.TM118.004156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Doucet A, Overall C (2008) Protease proteomics: revealing protease in vivo functions using systems biology approaches. Mol Asp Med 29:339–358. https://doi.org/10.1016/j.mam.2008.04.003

    Article  CAS  Google Scholar 

  4. Doucet A, Kleifeld O, Kizhakkedathu JN, Overall CM (2011) Identification of proteolytic products and natural protein N-termini by terminal amine isotopic labeling of substrates (TAILS). In: Gevaert K, Vandekerckhove J (eds) Gel-free proteomics, methods in molecular biology. Humana Press, Totowa, pp 273–287. https://doi.org/10.1007/978-1-61779-148-2_18

    Chapter  Google Scholar 

  5. Huesgen PF, Overall CM (2012) N- and C-terminal degradomics: new approaches to reveal biological roles for plant proteases from substrate identification. Physiol Plant 145:5–17. https://doi.org/10.1111/j.1399-3054.2011.01536.x

    Article  CAS  PubMed  Google Scholar 

  6. Kaushal P, Lee C (2021) N-terminomics – its past and recent advancements. J Proteome 233:104089. https://doi.org/10.1016/j.jprot.2020.104089

    Article  CAS  Google Scholar 

  7. Kleifeld O, Doucet A, auf dem Keller U, Prudova A, Schilling O, Kainthan RK, Starr AE, Foster LJ, Kizhakkedathu JN, Overall CM (2010) Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat Biotechnol 28:281–288. https://doi.org/10.1038/nbt.1611

    Article  CAS  PubMed  Google Scholar 

  8. Kleifeld O, Doucet A, Prudova A, auf dem Keller U, Gioia M, Kizhakkedathu JN, Overall CM (2011) Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. Nat Protoc 6:1578–1611. https://doi.org/10.1038/nprot.2011.382

    Article  CAS  PubMed  Google Scholar 

  9. López-Otín C, Bond JS (2008) Proteases: multifunctional enzymes in life and disease*. J Biol Chem 283:30433–30437. https://doi.org/10.1074/jbc.R800035200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Madzharova E, Sabino F, auf dem Keller U (2019) Exploring extracellular matrix degradomes by TMT-TAILS N-terminomics. In: Sagi I, Afratis NA (eds) Collagen, methods in molecular biology. Springer, New York, pp 115–126. https://doi.org/10.1007/978-1-4939-9095-5_8

    Chapter  Google Scholar 

  11. Mahrus S, Trinidad JC, Barkan DT, Sali A, Burlingame AL, Wells JA (2008) Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134:866–876. https://doi.org/10.1016/j.cell.2008.08.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. McDonald L, Robertson DHL, Hurst JL, Beynon RJ (2005) Positional proteomics: selective recovery and analysis of N-terminal proteolytic peptides. Nat Methods 2:955–957. https://doi.org/10.1038/nmeth811

    Article  CAS  PubMed  Google Scholar 

  13. Oveland E, Muth T, Rapp E, Martens L, Berven FS, Barsnes H (2015) Viewing the proteome: how to visualize proteomics data? Proteomics 15:1341–1355. https://doi.org/10.1002/pmic.201400412

    Article  CAS  PubMed  Google Scholar 

  14. PCAWG Drivers and Functional Interpretation Working Group, PCAWG Consortium, Paczkowska M, Barenboim J, Sintupisut N, Fox NS, Zhu H, Abd-Rabbo D, Mee MW, Boutros PC, Reimand J (2020) Integrative pathway enrichment analysis of multivariate omics data. Nat Commun 11:735. https://doi.org/10.1038/s41467-019-13983-9

    Article  CAS  Google Scholar 

  15. Prudova A, Gocheva V, auf dem Keller U, Eckhard U, Olson OC, Akkari L, Butler GS, Fortelny N, Lange PF, Mark JC, Joyce JA, Overall CM (2016) TAILS N-terminomics and proteomics show protein degradation dominates over proteolytic processing by cathepsins in pancreatic tumors. Cell Rep 16:1762–1773. https://doi.org/10.1016/j.celrep.2016.06.086

    Article  CAS  PubMed  Google Scholar 

  16. Puente XS, Sánchez LM, Overall CM, López-Otín C (2003) Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 4:544–558. https://doi.org/10.1038/nrg1111

    Article  CAS  PubMed  Google Scholar 

  17. Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD (2018) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46:D624–D632. https://doi.org/10.1093/nar/gkx1134

    Article  CAS  PubMed  Google Scholar 

  18. Ree R, Varland S, Arnesen T (2018) Spotlight on protein N-terminal acetylation. Exp Mol Med 50:1–13. https://doi.org/10.1038/s12276-018-0116-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sabino F, Hermes O, Egli FE, Kockmann T, Schlage P, Croizat P, Kizhakkedathu JN, Smola H, auf dem Keller U (2015) In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates. Mol Cell Proteomics 14:354–370. https://doi.org/10.1074/mcp.M114.043414

    Article  CAS  PubMed  Google Scholar 

  20. Schlage P, Egli FE, Nanni P, Wang LW, Kizhakkedathu JN, Apte SS, auf dem Keller U (2014) Time-resolved analysis of the matrix metalloproteinase 10 substrate degradome. Mol Cell Proteomics 13:580–593. https://doi.org/10.1074/mcp.M113.035139

    Article  CAS  PubMed  Google Scholar 

  21. Staes A, Impens F, Van Damme P, Ruttens B, Goethals M, Demol H, Timmerman E, Vandekerckhove J, Gevaert K (2011) Selecting protein N-terminal peptides by combined fractional diagonal chromatography. Nat Protoc 6:1130–1141. https://doi.org/10.1038/nprot.2011.355

    Article  CAS  PubMed  Google Scholar 

  22. The UniProt Consortium (2018) UniProt: the universal protein knowledgebase. Nucleic Acids Res 46:2699. https://doi.org/10.1093/nar/gky092

    Article  CAS  PubMed  Google Scholar 

  23. Tinnefeld V, Venne AS, Sickmann A, Zahedi RP (2017) Enrichment of cross-linked peptides using charge-based fractional diagonal chromatography (ChaFRADIC). J Proteome Res 16:459–469. https://doi.org/10.1021/acs.jproteome.6b00587

    Article  CAS  PubMed  Google Scholar 

  24. Venne AS, Solari FA, Faden F, Paretti T, Dissmeyer N, Zahedi RP (2015) An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana. Proteomics 15:2458–2469. https://doi.org/10.1002/pmic.201500014

    Article  CAS  PubMed  Google Scholar 

  25. Wang R, Wang G (2019) Protein modification and autophagy activation. Adv Exp Med Biol 1206:237–259. https://doi.org/10.1007/978-981-15-0602-4_12

    Article  CAS  PubMed  Google Scholar 

  26. Weeks AM, Byrnes JR, Lui I, Wells JA (2021) Mapping proteolytic neo-N termini at the surface of living cells. Proc Natl Acad Sci 118:e2018809118. https://doi.org/10.1073/pnas.2018809118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Weng SSH, Demir F, Ergin EK, Dirnberger S, Uzozie A, Tuscher D, Nierves L, Tsui J, Huesgen PF, Lange PF (2019) Sensitive determination of proteolytic proteoforms in limited microscale proteome samples. Mol Cell Proteomics 18:2335–2347. https://doi.org/10.1074/mcp.TIR119.001560

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wilson CH, Indarto D, Doucet A, Pogson LD, Pitman MR, McNicholas K, Menz RI, Overall CM, Abbott CA (2013) Identifying natural substrates for dipeptidyl peptidases 8 and 9 using terminal amine isotopic labeling of substrates (TAILS) reveals in vivo roles in cellular homeostasis and energy metabolism. J Biol Chem 288:13936–13949. https://doi.org/10.1074/jbc.M112.445841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark

    Konstantinos Kalogeropoulos, Louise Bundgaard & Ulrich auf dem Keller

Authors
  1. Konstantinos Kalogeropoulos
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  2. Louise Bundgaard
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  3. Ulrich auf dem Keller
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Correspondence to Ulrich auf dem Keller .

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  1. VIB-UGent Center for Medical Biotechnology, Ghent, Belgium

    Kris Gevaert

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Kalogeropoulos, K., Bundgaard, L., auf dem Keller, U. (2023). Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics. In: Gevaert, K. (eds) Mass Spectrometry-Based Proteomics. Methods in Molecular Biology, vol 2718. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3457-8_7

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  • DOI: https://doi.org/10.1007/978-1-0716-3457-8_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3456-1

  • Online ISBN: 978-1-0716-3457-8

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