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Targeted Quantitation of Acetylated Lysine Peptides by Selected Reaction Monitoring Mass Spectrometry

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Sirtuins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1077))

Abstract

Mass spectrometry (MS) allows for the large-scale identification of multiple peptide analytes in complex mixtures. However, the low abundance of acetylated peptides in the overall mixture requires an enrichment step. After enrichment, the resulting acetylated peptides of interest can be quantitated using selected reaction monitoring (SRM)-MS with stable isotope dilution. Here, we describe the enrichment of lysine acetylated peptides from typsin digested mouse liver mitochondria, and the targeted quantitation of a known lysine acetylation site in succinate dehydrogenase A using SRM-MS on a triple quadrupole instrument.

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References

  1. Chen Y, Zhao W, Yang JS, Cheng Z, Luo H, Lu Z, Tan M, Gu W, Zhao Y (2012) Quantitative acetylome analysis reveals the roles of SIRT1 in regulating diverse substrates and cellular pathways. Mol Cell Proteomics 11(10):1048–1062. doi:10.1074/mcp.M112.019547, [pii] M112. 019547

    Article  PubMed  CAS  Google Scholar 

  2. Schilling B, Rardin MJ, MacLean BX, Zawadzka AM, Frewen BE, Cusack MP, Sorensen DJ, Bereman MS, Jing E, Wu CC, Verdin E, Kahn CR, Maccoss MJ, Gibson BW (2012) Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation. Mol Cell Proteomics 11(5):202–214. doi:10.1074/mcp.M112.017707, [pii] M112.017707

    Article  PubMed  CAS  Google Scholar 

  3. Addona TA, Abbatiello SE, Schilling B, Skates SJ, Mani DR, Bunk DM, Spiegelman CH, Zimmerman LJ, Ham AJ, Keshishian H, Hall SC, Allen S, Blackman RK, Borchers CH, Buck C, Cardasis HL, Cusack MP, Dodder NG, Gibson BW, Held JM, Hiltke T, Jackson A, Johansen EB, Kinsinger CR, Li J, Mesri M, Neubert TA, Niles RK, Pulsipher TC, Ransohoff D, Rodriguez H, Rudnick PA, Smith D, Tabb DL, Tegeler TJ, Variyath AM, Vega-Montoto LJ, Wahlander A, Waldemarson S, Wang M, Whiteaker JR, Zhao L, Anderson NL, Fisher SJ, Liebler DC, Paulovich AG, Regnier FE, Tempst P, Carr SA (2009) Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat Biotechnol 27(7):633–641. doi:10.1038/nbt.1546, [pii] nbt.1546

    Article  PubMed  CAS  Google Scholar 

  4. Keshishian H, Addona T, Burgess M, Kuhn E, Carr SA (2007) Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics 6(12):2212–2229. doi:10.1074/mcp.M700354-MCP200, [pii] M700354-MCP200

    Article  PubMed  CAS  Google Scholar 

  5. Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100(12):6940–6945. doi:10.1073/pnas.0832254100, [pii] 0832254100

    Article  PubMed  CAS  Google Scholar 

  6. Rardin MJ, Taylor GS, Dixon JE (2009) Distinguishing mitochondrial inner membrane orientation of dual specific phosphatase 18 and 21. Methods Enzymol 457:275–287. doi:10.1016/S0076-6879(09)05015-0, [pii] S0076-6879(09)05015-0

    PubMed  CAS  Google Scholar 

  7. Shrivastava A, Gupta VB (2011) Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young Sci 2(1):21–25. doi:10.4103/2229-5186.79345

    Article  Google Scholar 

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Acknowledgments

This work was supported by NIH grant R24 DK085610 (B.W.G.) and the NCRR shared instrumentation grant S10 RR027953 (B.W.G.) for the 5500 QTRAP system.

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Authors and Affiliations

  1. Buck Institute for Research on Aging, Novato, CA, USA

    Matthew J. Rardin, Jason M. Held & Bradford W. Gibson

Authors
  1. Matthew J. Rardin
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  2. Jason M. Held
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  3. Bradford W. Gibson
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Editor information

Editors and Affiliations

  1. Duke University Medical Center Institute of Molecular Physiology, Durham, North Carolina, USA

    Matthew D. Hirschey

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Rardin, M.J., Held, J.M., Gibson, B.W. (2013). Targeted Quantitation of Acetylated Lysine Peptides by Selected Reaction Monitoring Mass Spectrometry. 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_8

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  • DOI: https://doi.org/10.1007/978-1-62703-637-5_8

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-636-8

  • Online ISBN: 978-1-62703-637-5

  • eBook Packages: Springer Protocols

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