Abstract
Proteins directly control almost all cellular processes and researchers in many biological areas routinely use mass spectrometry for the characterization of proteins. Amongst a growing list of available quantitative proteomic techniques, Stable Isotope Labeling by Amino acids in Culture (SILAC) remains one of the most simple, accurate, and robust techniques for cultured cellular systems. SILAC enables strict quantitative peptide measurements, thus removing false positives and facilitates large-scale kinetics of entire proteomes. In this, chapter we describe an optimized labeling strategy and experimental design for SILAC workflows for characterizing the components downstream of cell death stimuli.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Strasser A, O’Connor L, Dixit VM (2000) Apoptosis signaling. Annu Rev Biochem 69(1):217–245
Chowdhury I, Tharakan B, Bhat GK (2008) Caspases—an update. Comp Biochem Physiol B Biochem Mol Biol 151(1):10–27
Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR, Vandekerckhove J (2003) Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat Biotechnol 21:566–569
Van Damme P, Martens L, Van Damme J, Hugelier K, Staes A, Vandekerckhove J, Gevaert K (2005) Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nat Methods 2:771–777
Staes A, Van Damme P, Helsens K, Demol H, Vandekerckhove J, Gevaert K (2008) Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC). Proteomics 8:1362–1370
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
Impens F, Colaert N, Helsens K, Ghesquiere B, Timmerman E, De Bock PJ, Chain BM, Vandekerckhove J, Gevaert K (2010) A quantitative proteomics design for systematic identification of protease cleavage events. Mol Cell Proteomics 9:2327–2333
Impens F, Colaert N, Helsens K, Plasman K, Van Damme P, Vandekerckhove J, Gevaert K (2010) MS-driven protease substrate degradomics. Proteomics 10:1284–1296
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
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
Thiede B, Dimmler C, Siejak F, Rudel T (2001) Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis. J Biol Chem 276:26044–26050
Thiede B, Siejak F, Dimmler C, Rudel T (2002) Prediction of translocation and cleavage of heterogeneous ribonuclear proteins and Rho guanine nucleotide dissociation inhibitor 2 during apoptosis by subcellular proteome analysis. Proteomics 2:996–1006
Agard NJ, Wells JA (2009) Methods for the proteomic identification of protease substrates. Curr Opin Chem Biol 13:503–509
Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422(6928):198–207
Steen H, Mann M (2004) The ABC’s (and XYZ’s) of peptide sequencing. Nat Rev Mol Cell Biol 5(9):699–711
Medzihradszky KF (2005) Peptide sequence analysis. Methods Enzymol 402:209–244
Hildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P, Pierotti C, Garnier JM, Dobson RC, Webb AI, Tripaydonis A, Babon JJ, Mulcair MD, Scanlon MJ, Alexander WS, Wilks AF, Czabotar PE, Lessene G, Murphy JM, Silke J (2014) Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc Natl Acad Sci 111(42):15072–15077
Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, Mann M (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4(5):698–705
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Webb, A.I. (2016). Proteomic Profiling of Cell Death: Stable Isotope Labeling and Mass Spectrometry Analysis. In: Puthalakath, H., Hawkins, C. (eds) Programmed Cell Death. Methods in Molecular Biology, vol 1419. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3581-9_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3581-9_20
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3579-6
Online ISBN: 978-1-4939-3581-9
eBook Packages: Springer Protocols