Abstract
Improved sample preparation techniques and increasingly sensitive mass spectrometry (MS) analysis have revolutionized the study of protein post-translational modifications (PTMs) (Rush et al., Nat Biotechnol 23:94–101, 2005). Here, we describe a general approach for immunopurification and MS-based identification of acetylated proteins in biological samples. This approach is useful to characterize changes in the acetylome in response to biological interventions (Schwer et al., Aging Cell 8:604–606, 2009).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Schwer B, Eckersdorff M, Li Y, Silva JC, Fermin D, Kurtev MV, Giallourakis C, Comb MJ, Alt FW, Lombard DB (2009) Calorie restriction alters mitochondrial protein acetylation. Aging Cell 8(5):604–606. doi:ACE503 [pii] 10.1111/j.1474-9726.2009.00503.x
Norvell A, McMahon SB (2010) Cell biology. Rise of the rival. Science 327(5968):964–965. doi:327/5968/964 [pii] 10.1126/science.1187159
Baur JA, Ungvari Z, Minor RK, Le Couteur DG, de Cabo R (2012) Are sirtuins viable targets for improving healthspan and lifespan? Nat Rev Drug Discov 11(6):443–461. doi:10.1038/nrd3738 nrd3738 [pii]
Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325(5942):834–840. doi:1175371 [pii] 10.1126/science.1175371
Zhao S, Xu W, Jiang W, Yu W, Lin Y, Zhang T, Yao J, Zhou L, Zeng Y, Li H, Li Y, Shi J, An W, Hancock SM, He F, Qin L, Chin J, Yang P, Chen X, Lei Q, Xiong Y, Guan KL (2010) Regulation of cellular metabolism by protein lysine acetylation. Science 327(5968):1000–1004. doi:327/5968/1000 [pii] 10.1126/science.1179689
Kendrick AA, Choudhury M, Rahman SM, McCurdy CE, Friederich M, Van Hove JL, Watson PA, Birdsey N, Bao J, Gius D, Sack MN, Jing E, Kahn CR, Friedman JE, Jonscher KR (2011) Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation. Biochem J 433(3):505–514. doi:BJ20100791 [pii] 10.1042/BJ20100791
Kim SC, Sprung R, Chen Y, Xu Y, Ball H, Pei J, Cheng T, Kho Y, Xiao H, Xiao L, Grishin NV, White M, Yang XJ, Zhao Y (2006) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23(4):607–618. doi:10.1016/j.molcel.2006.06.026
Ahn BH, Kim HS, Song S, Lee IH, Liu J, Vassilopoulos A, Deng CX, Finkel T (2008) A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci U S A 105(38):14447–14452. doi:0803790105 [pii] 10.1073/pnas.0803790105
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75(3):663–670
Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD, Comb MJ (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol 23(1):94–101. doi:10.1038/nbt1046
Acknowledgements
The authors would like to thank members of the Lombard lab for helpful discussions, and the PTMScan Service Group at Cell Signaling Technology (Matt P. Stokes, Charles L. Farnsworth, and Hongbo Gu) for their assistance in reviewing the protocol. Work in the Lombard lab is supported by NIH grants R01GM101171 and R21CA177925 (Lombard), R01HL114858 (Lukacs), DP3DK094292 (Brosius and others), and a New Scholar in Aging award from the Ellison Medical Foundation (Lombard).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Li, Y., Silva, J.C., Skinner, M.E., Lombard, D.B. (2013). Mass Spectrometry-Based Detection of Protein Acetylation. In: Hirschey, M. (eds) Sirtuins. Methods in Molecular Biology, vol 1077. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-637-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-62703-637-5_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-636-8
Online ISBN: 978-1-62703-637-5
eBook Packages: Springer Protocols