Protein S‐acylation, predominately in the form of palmitoylation, is a reversible lipid post‐translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S‐acylation have been extensively studied in mammals owing to remarkable development of high‐resolution proteomics and the discovery of the S‐acylation‐related enzymes. However, the advancement of S‐acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S‐acylation in
Human paraoxonase‐1 (PON1) is a high‐density lipoprotein‐associated enzyme with antioxidant, anti‐inflammatory, and antiapoptotic roles. The ability of PON1 to hydrolyze specific organophosphate (OP) compounds and prevent accumulation of oxidized lipids in lipoproteins has prompted a large number of studies investigating PON1's role in modulating toxicity and disease. Most of these studies, however, have only focused on
This article was corrected on 18 July 2022. See the end of the full text for details.
- NSF-PAR ID:
- 10238665
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 1
- Issue:
- 1
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Arabidopsis seedlings is described. The procedures are presented in detail, including preparation ofArabidopsis seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S‐acylated proteins/peptides based on the acyl‐biotin exchange method, and large‐scale identification of S‐acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S‐acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC.Basic Protocol 1 : Preparation ofArabidopsis seedling materialsBasic Protocol 2 : Isolation and enrichment of plasma membrane proteinsSupport Protocol 1 : Determination of protein concentration using BCA assayBasic Protocol 3 : Enrichment of S‐acylated proteins by acyl‐biotin exchange methodSupport Protocol 2 : Protein precipitation by methanol/chloroform methodBasic Protocol 4 : Trypsin digestion and proteomic analysisAlternate Protocol : Pre‐resin digestion and peptide‐level enrichment -
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Abstract Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein‐protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy‐to‐apply protocol for high‐stoichiometric, site‐specific labeling of a protein at its N‐terminus with
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