Abstract
Mass spectrometry (MS)-based methods typically assess acetylation by detection of a diagnostic ion at 126.1 m/z, corresponding to the immonium ion of acetyl-lysine –NH3, which is generated by collisionally induced dissociation. A novel implementation of this approach, based on the accurate mass and retention time technique, couples high mass resolution measurement with rapid cycling between low and elevated collision energies to generate intact and fragment high-resolution mass spectra. This allows acetyl lysine diagnostic ions at 126.1 m/z to be monitored and aligned to the precursor m/z based on retention time profile. The technique is termed Collisionally Induced Release of Acetyl Diagnostic. Sequence information is also obtained for acetylation site assignment. This technique to identify acetylation species is information independent as it does not require the sequence of the protein/peptides to identify acetylation, and thus complementary to data-dependent methods. It is suitable for analysis of acetylated peptides, or proteins enriched by immunoprecipitation with acetyl lysine-specific antibodies.
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References
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 325:834–840
Mischerikow N, Heck AJ (2011) Targeted large-scale analysis of protein acetylation. Proteomics 11:571–589
Borchers C, Parker CE, Deterding LJ, Tomer KB (1999) Preliminary comparison of precursor scans and liquid chromatography–tandem mass spectrometry on a hybrid quadrupole time-of-flight mass spectrometer. J Chromatogr A 854:119–130
Kim JY, Kim KW, Kwon HJ, Lee DW, Yoo JS (2002) Probing lysine acetylation with a modification specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation. Anal Chem 74:5443–5449
Dormeyer W, Ott M, Schnolzer M (2005) Probing lysine acetylation in proteins: strategies, limitations, and pitfalls of in vitro acetyl-transferase assays. Mol Cell Proteomics 4: 1226–1239
Domon B, Aebersold R (2006) Mass spectrometry and protein analysis. Science 312:212–217
Walther TC, Mann M (2010) Mass spectrometry-based proteomics in cell biology. J Cell Biol 190:491–500
Ow SY, Noirel J, Salim M, Evans C, Watson R, Wright PC (2010) Balancing robust quantification and identification for iTRAQ: application of UHR-ToF MS. Proteomics 10:2205–2213
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:551–567
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 23:607–618
Leech SH, Evans CA, Shaw L, Wong CH, Connolly J, Griffiths JR, Whetton AD, Corfe BM (2008) Proteomic analyses of intermediate filaments reveals cytokeratin8 is highly acetylated—implications for colorectal epithelial homeostasis. Proteomics 8:279–288
Yang XJ, Seto E (2008) Lysine acetylation: codified cross talk with other post translational modifications. Mol Cell 31:449–461
Unwin R, Griffiths J, Leverentz M, Grallert A, Hagan I, Whetton A (2005) Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity. Mol Cell Proteomics 4:1134–1144
Mollah S, Wertz IE, Phung Q, Arnott D, Dixit VM, Lill JR (2007) Targeted mass spectrometric strategy for global mapping of ubiquitination on proteins. Rapid Commun Mass Spectrom 21:3357–3364
Griffiths J, Unwin R, Evans C, Leech S, Corfe B, Whetton A (2007) The application of hypothesis driven strategy to the sensitive detection and location of acetylated lysine residues. JASMS 18:1423–1428
Lange V, Picotti P, Domon B, Aebersold R (2008) Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 4:222–235
Liu B, Lin Y, Darwanto A, Song X, Xu G, Zhang K (2009) Identification and characterization of propionylation at histone H3 lysine 23 in mammalian cells. J Biol Chem 284:32288–32295
Li GZ, Vissers JP, Silva JC, Golick D, Gorenstein MV, Geromanos SJ (2009) Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures. Proteomics 9(6):1696–1719
Acknowledgements
Financial support from the Engineering and Physical Sciences Research Council, ChELSI initiative EP/E036252/1 (CE, OSY, PCW), Cancer Research UK (DS), and Biochemical Biophysical Research Council (BMC). We thank the Bruker Daltonik GmbH research team, in particular Peter Sander, for provision and discussion on the Dissect algorithm.
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Evans, C.A., Ow, S.Y., Smith, D.L., Corfe, B.M., Wright, P.C. (2013). Application of the CIRAD Mass Spectrometry Approach for Lysine Acetylation Site Discovery. In: Hake, S., Janzen, C. (eds) Protein Acetylation. Methods in Molecular Biology, vol 981. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-305-3_2
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DOI: https://doi.org/10.1007/978-1-62703-305-3_2
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