Identification of newly translated thermo-sensitive proteins using pulse SILAC mass spectrometry and the GAL promoter system

• Published in: STAR protocols Vol. 4; no. 1; p. 102059
• Main Authors: Zhu, Mang, Calabrese, Gaetano, Wong, Ryan W.K., Mayor, Thibault
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-03-2023; Elsevier
• Subjects: High-throughput Screening; Mass Spectrometry; Model Organisms; Molecular Biology; Protein Biochemistry; Proteomics; Protocol; Systems biology; Protein Biochemistry; Molecular Biology; Systems biology; Mass Spectrometry; Proteomics; Model Organisms; High-throughput Screening
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102059

Some newly translated proteins are more susceptible to misfolding and aggregation upon heat shock in comparison to other proteins. To study these newly translated thermo-sensitive proteins on a proteomic scale, we present here a protocol that combines pulse-SILAC with biochemical fractionation for mass spectrometry analysis, followed by an orthogonal validation protocol for selected candidates using the GAL promoter system in Saccharomyces cerevisiae. This approach can be further developed to study other stresses and specific post-translational modifications or adapted to mammalian cells.
For complete details on the use and execution of this protocol, please refer to Zhu et al. (2022).1
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•Pulse-SILAC labeling to identify newly translated proteins by mass spectrometry•Detailed steps of yeast cryo-lysis and pellet fraction collection•Galactose induction for temporally defined protein expression in yeast
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Some newly translated proteins are more susceptible to misfolding and aggregation upon heat shock in comparison to other proteins. To study these newly translated thermo-sensitive proteins on a proteomic scale, we present here a protocol that combines pulse-SILAC with biochemical fractionation for mass spectrometry analysis, followed by an orthogonal validation protocol for selected candidates using the GAL promoter system in Saccharomyces cerevisiae. This approach can be further developed to study other stresses and specific post-translational modifications or adapted to mammalian cells.