Abstract
Cultured primary neurons are a well-established model for the study of neuronal function. Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires nearly complete metabolic labeling of proteins and therefore is difficult to apply to cultured primary neurons, which do not divide in culture. In a multiplex SILAC strategy, two different sets of heavy amino acids are used for labeling cells for the different experimental conditions. This allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. When combined with bioorthogonal noncanonical amino acid tagging (BONCAT), it allows for comparative proteomic analysis of de novo protein synthesis. Here we describe protocols that utilize the multiplex SILAC labeling strategy for primary cultured neurons to study steady-state and nascent proteomes.
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References
Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386
Ong SE, Mann M (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 1:2650–2660
Spellman DS, Deinhardt K, Darie CC et al (2008) Stable isotopic labeling by amino acids in cultured primary neurons: application to brain-derived neurotrophic factor-dependent phosphotyrosine-associated signaling. Mol Cell Proteomics 7:1067–1076
Liao L, Park SK, Xu T et al (2008) Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice. Proc Natl Acad Sci U S A 105:15281–15286
Elias JE, Haas W, Faherty BK et al (2005) Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations. Nat Methods 2:667–675
Selbach M, Schwanhausser B, Thierfelder N et al (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455:58–63
Zhang G, Deinhardt K, Chao MV et al (2011) Study of neurotrophin-3 signaling in primary cultured neurons using multiplex stable isotope labeling with amino acids in cell culture. J Proteome Res 10:2546–2554
Zhang G, Neubert TA, Jordan BA (2012) RNA binding proteins accumulate at the postsynaptic density with synaptic activity. J Neurosci 32:599–609
Zhang G, Bowling H, Hom N et al (2014) In-depth quantitative proteomic analysis of de novo protein synthesis induced by brain-derived neurotrophic factor. J Proteome Res 13:5707–5714
Bowling H, Bhattacharya A, Zhang G et al (2015) BONLAC: a combinatorial proteomic technique to measure stimulus-induced translational profiles in brain slices. Neuropharmacology 100:76–89
Van Hoof D, Pinkse MW, Oostwaard DW et al (2007) An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics. Nat Methods 4:677–678
Acknowledgments
This work was supported by NIH NINDS grant P30 NS050276 and NCRR grant S10 RR027990 to TAN.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Zhang, G., Deinhardt, K., Neubert, T.A. (2023). Stable Isotope Labeling by Amino Acids and Bioorthogonal Noncanonical Amino Acid Tagging in Cultured Primary Neurons. In: Luque-Garcia, J.L. (eds) SILAC. Methods in Molecular Biology, vol 2603. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2863-8_13
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DOI: https://doi.org/10.1007/978-1-0716-2863-8_13
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2862-1
Online ISBN: 978-1-0716-2863-8
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