Stable isotope labeling by Amino Acids and bioorthogonal noncanonical Amino Acid tagging in cultured primary neurons

Zhang, Guoan, Deinhardt, Katrin and Neubert, Thomas A. (2022) Stable isotope labeling by Amino Acids and bioorthogonal noncanonical Amino Acid tagging in cultured primary neurons. In, Methods in molecular biology (Clifton, N.J.). (SILAC, 2603) New York, NY. Humana, pp. 163-171. (doi:10.1007/978-1-0716-2863-8_13).

Record type: Book Section

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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SILAC AND BONLAC IN NEURONS_TN_GZ[70] - Accepted Manuscript

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