Abstract
Determination of drug targets and development of novel therapeutics for the treatment of different cancers are actively ongoing areas of research. Proteases being the second largest group of enzymes in humans present themselves as attractive targets for blocking and activation to treat malignancies. However, determination of the protease cleavage substrates is often missed by utilizing conventional modern proteomic approaches. The relatively low abundance of proteolytically processed, and mostly semi-tryptic, peptides compared to tryptic peptides generated in shotgun proteomics compounded with their poorer identification rates makes the identification of such critical peptides challenging and so are mostly overlooked. Our laboratory introduced Terminal Amine Isotopic Labeling of Substrates (TAILS) to identify N-terminal peptides from cleavage events. In this chapter we present a protocol from our complementary method carboxy-TAILS (C-TAILS) to identify C-terminal peptides in metabolically labeled cancer cell lines.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Krueger KE, Srivastava S (2006) Posttranslational protein modifications: current implications for cancer detection, prevention, and therapeutics. Mol Cell Proteomics 5(10):1799–1810. https://doi.org/10.1074/mcp.R600009-MCP200
Burotto M, Chiou VL, Lee JM et al (2014) The MAPK pathway across different malignancies: a new perspective. Cancer 120(22):3446–3456. https://doi.org/10.1002/cncr.28864
Dennis JW, Laferte S, Waghorne C et al (1987) Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 236(4801):582–585
Ma X, Dong W, Su Z et al (2016) Functional roles of sialylation in breast cancer progression through miR-26a/26b targeting ST8SIA4. Cell Death Dis 7(12):e2561. https://doi.org/10.1038/cddis.2016.427
Puente XS, Sanchez LM, Overall CM et al (2003) Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 4(7):544–558. https://doi.org/10.1038/nrg1111
Fortelny N, Cox JH, Kappelhoff R et al (2014) Network analyses reveal pervasive functional regulation between proteases in the human protease web. PLoS Biol 12(5):e1001869. https://doi.org/10.1371/journal.pbio.1001869
Overall CM, Blobel CP (2007) In search of partners: linking extracellular proteases to substrates. Nat Rev Mol Cell Biol 8(3):245–257. https://doi.org/10.1038/nrm2120
Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422(6928):198–207. https://doi.org/10.1038/nature01511
Hebert AS, Richards AL, Bailey DJ et al (2014) The one hour yeast proteome. Mol Cell Proteomics 13(1):339–347. https://doi.org/10.1074/mcp.M113.034769
Steen H, Mann M (2004) The ABC's (and XYZ's) of peptide sequencing. Nat Rev Mol Cell Biol 5(9):699–711. https://doi.org/10.1038/nrm1468
Solis N, Overall CM (2016) Mass spectrometry-based methodologies for studying proteolytic networks and the degradome. In: Bradshaw RA, Stahl PD (eds) Encyclopedia of cell biology, vol 1. Elsevier, Waltham MA, pp 568–581
Kleifeld O, Doucet A, auf dem Keller U et al (2010) Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat Biotechnol 28(3):281–288. https://doi.org/10.1038/nbt.1611
Kleifeld O, Doucet A, Prudova A et al (2011) Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. Nat Protoc 6(10):1578–1611. https://doi.org/10.1038/nprot.2011.382
Schilling O, Barre O, Huesgen PF et al (2010) Proteome-wide analysis of protein carboxy termini: C terminomics. Nat Methods 7(7):508–511. https://doi.org/10.1038/nmeth.1467
Zhang Y, He Q, Ye J et al (2015) Systematic optimization of C-terminal amine-based isotope labeling of substrates approach for deep screening of C-Terminome. Anal Chem 87(20):10354–10361. https://doi.org/10.1021/acs.analchem.5b02451
Somasundaram P, Koudelka T, Linke D et al (2016) C-terminal charge-reversal Derivatization and parallel use of multiple proteases facilitates identification of protein C-termini by C-Terminomics. J Proteome Res 15(4):1369–1378. https://doi.org/10.1021/acs.jproteome.6b00146
Huesgen PF, Lange PF, Rogers LD et al (2015) LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification. Nat Methods 12(1):55–58. https://doi.org/10.1038/nmeth.3177
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. https://doi.org/10.1038/nbt.1511
Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138(1):141–143
Geiger T, Cox J, Ostasiewicz P et al (2010) Super-SILAC mix for quantitative proteomics of human tumor tissue. Nat Methods 7(5):383–385. https://doi.org/10.1038/nmeth.1446
Good DM, Wirtala M, McAlister GC et al (2007) Performance characteristics of electron transfer dissociation mass spectrometry. Mol Cell Proteomics 6(11):1942–1951. https://doi.org/10.1074/mcp.M700073-MCP200
Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11(12):2301–2319. https://doi.org/10.1038/nprot.2016.136
Acknowledgments
N.S. is funded by the Michael Smith Foundation for Health Research of Canada (Grant ID:16642) and by the National Health and Medical Research Council of Australia (APP1126842). C.M.O holds a Canada Research Chair in Protease Proteomics and Systems Biology.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Solis, N., Overall, C.M. (2018). Identification of Protease Cleavage Sites and Substrates in Cancer by Carboxy-TAILS (C-TAILS). In: Cal, S., Obaya, A. (eds) Proteases and Cancer. Methods in Molecular Biology, vol 1731. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7595-2_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7595-2_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7594-5
Online ISBN: 978-1-4939-7595-2
eBook Packages: Springer Protocols