Mass‐Tag Labeling Using Acyl‐PEG Exchange for the Determination of Endogenous Protein S‐Fatty Acylation

Avital Percher1, Emmanuelle Thinon1, Howard Hang1

1 Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.17
DOI:  10.1002/cpps.36
Online Posting Date:  August, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The covalent coupling of fatty acids to proteins provides an important mechanism of regulation in cells. In eukaryotes, cysteine fatty acylation (S‐fatty acylation) has been shown to be critical for protein function in a variety of cellular pathways as well as microbial pathogenesis. While methods developed over the past decade have improved the detection and profiling of S‐fatty acylation, these are hampered in their ability to characterize endogenous protein S‐fatty acylation levels under physiological conditions. Furthermore, understanding the contribution of specific sites and levels of S‐fatty acylation remains a major challenge. To evaluate S‐fatty acylation of endogenous proteins as well as to determine the number of S‐fatty acylation events, we developed the acyl‐PEG exchange (APE) that utilizes cysteine‐specific chemistry to exchange S‐fatty acylation sites with mass‐tags of defined size, which can be readily observed by western blotting. APE provides a readily accessible approach to investigate endogenous S‐fatty acylation from any sample source, with high sensitivity and broad applicability that complements the current toolbox of methods for thioester‐based post‐translational modifications. © 2017 by John Wiley & Sons, Inc.

Keywords: mass‐shift; PEGylation; post‐translational modification quantification; s‐fatty‐acylation

PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1:

  • Phosphate‐buffered saline (PBS; appendix 2E), sterile
  • 1× lysis buffer (see recipe)
  • 0.5 M EDTA, pH 8 (Sigma)
  • Bicinchoninic acid (BCA) assay kit (unit 3.4; Olson and Markwell, )
  • 200 mM neutralized TCEP (see recipe)
  • 1 M N‐ethyl maleimide (NEM; see recipe)
  • Methanol, pre‐chilled on ice
  • Chloroform, pre‐chilled on ice
  • 1× TEA buffer/4% SDS (see recipe)
  • 1× TEA buffer/4% SDS (see recipe) with 4 mM EDTA
  • 1 M neutralized hydroxylamine hydrochloride (NH 2OH) (see recipe)
  • 1× TEA buffer/0.2% Triton X‐100 (see recipe)
  • 5 and 10 kDa methoxypolyethylene glycol maleimide (mPEG‐Mal; see reciperecipes)
  • 1× and 4× Laemmli buffer (see recipe)
  • Anti‐calnexin rabbit polyclonal primary antibody; dilute 1:2,000; Abcam, cat. no. ab22595
  • Goat anti‐rabbit secondary antibody; dilute 1:5000; Calbiochem, cat. no. DC03L
  • 1.5‐ml microcentrifuge tubes (1.5 ml)
  • Refrigerated microcentrifuge
  • Sonicator (e.g., Ultrasonic Cleaner, VWR)
  • Nutating mixer (e.g., Thomas Scientific)
  • 95oC heating block
  • Vacuum evaporator (e.g., Centrivap Concentrator, Labconco)
  • 4–20% Criterion‐TGX Stain Free polyacrylamide gels (Bio‐Rad)
  • Nitrocellulose membrane (0.2 µm)
  • Additional reagents and equipment for trypsinization ( appendix 3C; Phelan, ), BCA protein assay (unit 3.4; Olson and Markwell, ), SDS‐PAGE (unit 10.1; Gallagher, ), and western blotting [unit 10.7 (Goldman, Ursitti, Mozdzanowski, & Speicher, ) and unit 10.8 (Goldman, Harper, & Speicher, )]
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Brett, K., Kordyukova, L. V., Serebryakova, M. V., Mintaev, R. R., Alexeevski, A. V., & Veit, M. (2014). Site‐specific S‐acylation of influenza virus hemagglutinin: The location of the acylation site relative to the membrane border is the decisive factor for attachment of stearate. The Journal of Biological Chemistry, 289(50), 34978–34989. doi: 10.1074/jbc.M114.586180.
  Caballero, M. C., Alonso, A. M., Deng, B., Attias, M., de Souza, W., & Corvi, M. M. (2016). Identification of new palmitoylated proteins in Toxoplasma gondii. Biochimica et Biophysica Acta, 1864(4), 400–408. doi: 10.1016/j.bbapap.2016.01.010.
  Chamberlain, L. H., & Shipston, M. J. (2015). The physiology of protein S‐acylation. Physiological Reviews, 95(2), 341–376. doi: 10.1152/physrev.00032.2014.
  Charron, G., Zhang, M. M., Yount, J. S., Wilson, J., Raghavan, A. S., Shamir, E., & Hang, H. C. (2009). Robust fluorescent detection of protein fatty‐acylation with chemical reporters. Journal of the American Chemical Society, 131(13), 4967–4975. doi: 10.1021/ja810122f.
  Chesarino, N. M., Hach, J. C., Chen, J. L., Zaro, B. W., Rajaram, M. V., Turner, J., … Yount, J. S. (2014). Chemoproteomics reveals Toll‐like receptor fatty acylation. BMC Biology, 12, 91. doi: 10.1186/s12915‐014‐0091‐3.
  Forrester, M. T., Hess, D. T., Thompson, J. W., Hultman, R., Moseley, M. A., Stamler, J. S., & Casey, P. J. (2011). Site‐specific analysis of protein S‐acylation by resin‐assisted capture. Journal of Lipid Research, 52(2), 393–398. doi: 10.1194/jlr.D011106.
  Fukata, Y., & Fukata, M. (2010). Protein palmitoylation in neuronal development and synaptic plasticity. Nature Reviews Neuroscience, 11(3), 161–175. doi: 10.1038/nrn2788.
  Gallagher, S. R. (2012). One‐dimensional SDS gel electrophoresis of proteins. Current Protocols in Protein Science, 68, 10.1.1–10.1.44. doi: 10.1002/0471140864.ps1001s68.
  Goldman, A., Harper, S., & Speicher, D. W. (2016). Detection of proteins on blot membranes. Current Protocols in Protein Science, 86, 10.8.1‐10.8.11. doi: 10.1002/cpps.15.
  Goldman, A., Ursitti, J. A., Mozdzanowski, J., & Speicher, D. W. (2015). Electroblotting from polyacrylamide gels. Current Protocols in Protein Science, 82, 10.7.1‐10.7.16. doi: 10.1002/0471140864.ps1007s82.
  Hang, H. C., Wilson, J. P., & Charron, G. (2011). Bioorthogonal chemical reporters for analyzing protein lipidation and lipid trafficking. Accounts of Chemical Research, 44(9), 699–708. doi: 10.1021/ar200063v.
  Henkel, M., Rockendorf, N., & Frey, A. (2016). Selective and efficient cysteine conjugation by maleimides in the presence of phosphine reductants. Bioconjugate Chemistry, 27(10), 2260–2265. doi: 10.1021/acs.bioconjchem.6b00371.
  Ji, Y., Leymarie, N., Haeussler, D. J., Bachschmid, M. M., Costello, C. E., & Lin, C. (2013). Direct detection of S‐palmitoylation by mass spectrometry. Analytical Chemistry, 85(24), 11952–11959. doi: 10.1021/ac402850s.
  Kang, R., Wan, J., Arstikaitis, P., Takahashi, H., Huang, K., Bailey, A. O., … El‐Husseini, A. (2008). Neural palmitoyl‐proteomics reveals dynamic synaptic palmitoylation. Nature, 456(7224), 904–909. doi: 10.1038/nature07605.
  Kantner, T., & Watts, A. G. (2016). Characterization of reactions between water‐soluble trialkylphosphines and thiol alkylating reagents: Implications for protein‐conjugation reactions. Bioconjugate Chemistry, 27(10), 2400–2406. doi: 10.1021/acs.bioconjchem.6b00375.
  Liang, X., Nazarian, A., Erdjument‐Bromage, H., Bornmann, W., Tempst, P., & Resh, M. D. (2001). Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction. The Journal of Biological Chemistry, 276(33), 30987–30994. doi: 10.1074/jbc.M104018200.
  Lin, D. T., & Conibear, E. (2015a). ABHD17 proteins are novel protein depalmitoylases that regulate N‐Ras palmitate turnover and subcellular localization. Elife, 4, e11306. doi: 10.7554/eLife.11306.
  Lin, D. T., & Conibear, E. (2015b). Enzymatic protein depalmitoylation by acyl protein thioesterases. Biochemical Society Transactions, 43(2), 193–198. doi: 10.1042/BST20140235.
  Linder, M. E., & Deschenes, R. J. (2007). Palmitoylation: Policing protein stability and traffic. Nature Reviews. Molecular Cell Biology, 8(1), 74–84. doi: 10.1038/nrm2084.
  Muszbek, L., Haramura, G., Cluette‐Brown, J. E., Van Cott, E. M., & Laposata, M. (1999). The pool of fatty acids covalently bound to platelet proteins by thioester linkages can be altered by exogenously supplied fatty acids. Lipids, 34(Suppl.), S331–337. doi: 10.1007/BF02562334.
  Olson, B. J., & Markwell, J. (2007). Assays for determination of protein concentration. Current Protocols in Protein Science, 48, 3.4.1–3.4.29. doi: 10.1002/0471140864.ps0304s48.
  Peng, T., Thinon, E., & Hang, H. C. (2016). Proteomic analysis of fatty‐acylated proteins. Current Opinion in Chemical Biology, 30, 77–86. doi: 10.1016/j.cbpa.2015.11.008.
  Percher, A., Ramakrishnan, S., Thinon, E., Yuan, X., Yount, J. S., & Hang, H. C. (2016). Mass‐tag labeling reveals site‐specific and endogenous levels of protein S‐fatty acylation. Proceedings of the National Academy of Sciences of the United States of America, 113(16), 4302–4307. doi: 10.1073/pnas.1602244113.
  Phelan, M. C. (2006). Techniques for mammalian cell tissue culture. Current Protocols in Protein Science, 46, A.3C.1–A.3C.18.
  Roberts, B. J., Svoboda, R. A., Overmiller, A. M., Lewis, J. D., Kowalczyk, A. P., Mahoney, M. G., … Wahl, J. K., 3rd. (2016). Palmitoylation of desmoglein 2 is a regulator of assembly dynamics and protein turnover. The Journal of Biological Chemistry, 291(48), 24857–24865. doi: 10.1074/jbc.M116.739458.
  Thinon, E., Percher, A., & Hang, H. C. (2016). Bioorthogonal chemical reporters for monitoring unsaturated fatty‐acylated proteins. Chembiochem, 17(19), 1800–1803. doi: 10.1002/cbic.201600213.
  Wan, J., Roth, A. F., Bailey, A. O., & Davis, N. G. (2007). Palmitoylated proteins: Purification and identification. Nature Protocols, 2(7), 1573–1584. doi: 10.1038/nprot.2007.225.
  Westcott, N.P., Fernandez, J. P., Molina, H., & Hang, H.C. (2017). Chemical proteomics reveals ADP‐ribosylation of small GTPases during oxidative stress. Nature Chemical Biology, 13(3):302–308. doi: 10.1038/nchembio.2280.
  Yokoi, N., Fukata, Y., Sekiya, A., Murakami, T., Kobayashi, K., & Fukata, M. (2016). Identification of PSD‐95 depalmitoylating enzymes. Journal of Neuroscience, 36(24), 6431–6444. doi: 10.1523/JNEUROSCI.0419‐16.2016.
PDF or HTML at Wiley Online Library