Mechanisms and functions of protein S-acylation.

Proteins can be modified by lipids in various ways, for example, by myristoylation, palmitoylation, farnesylation, and geranylgeranylation-these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analysis of endogenous protein N-terminal myristoylation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore myristoylation sites in HeLa cells and identified a total of 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence myristoylated proteins identified by myristic acid analog-based chemical proteomics. Isolation of myristoylated peptides from HeLa digests prepared with different proteases enabled the identification of different myristoylated sites, extending the coverage of N-myristoylome. Finally, we analyzed in vivo myristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of protein N-terminal myristoylation. 

Application of liquid-liquid extraction for N-terminal myristoylation proteomics.

Protein S-acylation is an improtant lipid modification characteristic of heterogeneity in acyl chain and dynamicity in acylation/deacylation cycling. Most S-acylproteomic research was limited by indirect identification via modified proteins/peptides without...

Simultaneous and Site-specific Profiling of Heterogeneity and Turnover in Protein S-acylation by Intact S-acylated Peptides Analysis with a Cleavable Bioorthogonal Tag

N-myristoylation is one of the most widespread and important lipidation in eukaryotes and some prokaryotes, which is formed by covalently attaching various fatty acids (predominantly myristic acid C14:0) to the N-terminal glycine of proteins. Disorder of N-myristoylation is critically implicated in numerous physiological and pathological processes. Here, we presented a method for purification and comprehensive characterization of endogenous, intact N-glycine lipid-acylated peptides, which combined the negative selection method for N-terminome and the nanographite fluoride-based solid-phase extraction method (NeS-nGF SPE). After optimizing experimental conditions, we conducted the first global profiling of the endogenous and heterogeneous modification states for N-terminal glycine, pinpointing the precise sites and their associated lipid moieties. Totally, we obtained 76 N-glycine lipid-acylated peptides, including 51 peptides with myristate (C14:0), 10 with myristoleate (C14:1), 6 with tetradecadienoicate (C14:2), 5 with laurate (C12:0) and 4 with lauroleate (C12:1). Therefore, our proteomic methodology could significantly facilitate precise and in-depth analysis of the endogenous N-myristoylome and its heterogeneity. 

Global analysis of N-myristoylation and its heterogeneity by combining N-terminomics and nanographite fluoride-based solid-phase extraction.

Protein lipidation is a common co- or post-translational modification that plays a crucial role in regulating the localization, interaction, and function of cellular proteins. Dysregulation of lipid modifications can lead to various diseases, including cancer, neurodegenerative diseases, and infectious diseases. Therefore, the identification of proteins undergoing lipidation and their lipidation sites should provide insights into many aspects of lipid biology, as well as providing potential targets for therapeutic strategies. Bottom-up proteomics using liquid chromatography/tandem mass spectrometry is a powerful technique for the global analysis of protein lipidation. Here, we review proteomic methods for profiling protein lipidation, focusing on the two major approaches: the use of chemical probes, such as lipid alkyne probes, and the use of enrichment techniques for endogenous lipid-modified peptides. The challenges facing these methods and the prospects for developing them further to achieve a comprehensive analysis of lipid modifications are discussed.

Exploring protein lipidation by mass spectrometry-based proteomics.

S-acylation is a widespread lipidation form in eukaryotes in which various fatty acids can be covalently attached to specific cysteine residues. However, due to the low reactivity of the lipid moieties and lack of specific antibodies, purification of intact S-acylated peptides remains challenging. Here, we developed a pretreatment method for direct separation and global analysis of endogenously intact S-acylated peptides by nanographite fluoride-based solid-phase extraction (nGF-SPE), together with the investigation and optimization of the enrichment procedure as well as the LC-MS/MS analysis process. Consequently, we performed the first global profiling of endogenously intact S-acylated peptides, with 701 S-palmitoylated peptides from HeLa cell lysates in a restricted search. Furthermore, coupling the nGF-SPE method with open search mode, altogether 1119 intact S-acylated peptides were identified with the attached palmitate, palmitoleate, myristate, and octanoate chain, respectively, providing a global insight into the endogenously heterogeneous modification state. Notably, we found and validated that S-palmitoleoylation (C16:1) provided less affinity toward lipid rafts compared with S-palmitoylation (C16:0). This study developed the first straightforward way to characterize endogenously intact S-acylated peptides on a proteome-wide scale, providing the modified residues together with their attached lipid moieties simultaneously, which paves the way for further understanding of protein S-acylation. 

Global Analysis of Endogenously Intact S-Acylated Peptides Reveals Localization Differentiation of Heterogeneous Lipid Chains in Mammalian Cells.

Protein post-translational modifications (PTMs), which include covalent addition of functional groups to proteins and removal of specific amino acid(s) from proteins after synthesis, are often regulated by enzymes and linked to various cellular processes. Identification of enzyme substrates and their modification sites can assist in understanding the control of PTM enzymes on cellular processes, which is important for comprehending physiopathological processes. Due to the rapid advancement of analytical techniques, mass spectrometry (MS)-based proteomic approach has been one of the dominant methods for detecting enzyme-substrate interactions and modification site preferences in biological samples. Here, the development and optimization of MS-based proteomic methodologies in profiling substrates of PTM enzymes are briefly reviewed. 

Mass spectrometry-based candidate substrate and site identification of PTM enzymes

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

Roujun Wu

Author Tools

Chat about Author

Papers

Global Analysis of Endogenously Intact S-Acylated Peptides Reveals Localization Differentiation of Heterogeneous Lipid Chains in Mammalian Cells.

Simultaneous and Site-specific Profiling of Heterogeneity and Turnover in Protein S-acylation by Intact S-acylated Peptides Analysis with a Cleavable Bioorthogonal Tag

Mass spectrometry-based candidate substrate and site identification of PTM enzymes