How to Perform Functional Annotation of Acylated Proteins and Analyze Their Associated Signaling Pathways?

Functional annotation and signaling pathway analysis of acylated proteins are essential for understanding their roles in cellular processes, metabolic regulation, and disease mechanisms.

What Are Acylated Proteins? Why Should They Be Studied?

Acylation represents an important class of post-translational protein modifications, with common types including:

• Acetylation

• Propionylation

• Butyrylation

• Succinylation

• Malonylation

• Palmitoylation, among others.

These modifications typically occur on lysine or cysteine residues and regulate protein stability, enzymatic activity, subcellular localization, and protein–protein interactions. They serve as critical regulatory factors in diverse biological processes such as signal transduction, metabolic regulation, and chromatin remodeling.

Strategies for Functional Annotation of Acylated Proteins

1. Function Prediction Based on Annotation Databases

Based on the list of acylated proteins derived from enrichment analysis, the following databases can be used for functional annotation:

• UniProt: provides fundamental functional and structural annotations of proteins.

• Gene Ontology (GO): describes protein functions from three aspects, including biological process (BP), molecular function (MF), and cellular component (CC).

• InterPro / Pfam: identifies functional domains and protein families.

• Swiss-Prot Keywords: supplies functional keywords associated with proteins.

Recommended tools:

• DAVID

• eggNOG-mapper

• g:Profiler

2. Focusing on the Biological Significance of Modification Sites

The functional consequences of protein acylation depend not only on the overall function of the target protein but also critically on the specific modification sites. Therefore, it is recommended to:

• Identify highly conserved sites (e.g., sites conserved across multiple species or enriched within key structural domains).

• Use established databases (such as PhosphoSitePlus and PLMD) to predict the biological functions of acylation sites.

• Integrate structural information (such as AlphaFold structural models) to analyze the potential roles of modification sites within the three-dimensional conformation of proteins.

Signaling Pathway Enrichment Analysis of Acylated Proteins

1. Pathway Analysis Based on KEGG / Reactome

Using the KEGG or Reactome databases, pathway enrichment analysis can be performed to identify the signaling pathways in which acylated proteins are involved, such as:

• Glycolysis / Tricarboxylic acid cycle / Fatty acid metabolism (metabolic pathways)

• mTOR / AMPK / NF-κB / MAPK (signal transduction pathways)

• Chromatin modification and epigenetic regulation (e.g., HDAC/SIRT pathways)

This facilitates the inference of the potential roles of acylation in regulating metabolic states, inflammatory responses, and cell fate decisions.

Recommended tools:

• ClusterProfiler (R package): supports enrichment analysis of KEGG, Reactome, and WikiPathways and is suitable for high-throughput datasets

• Metascape: an integrated visualization platform for GO, KEGG, Reactome, and PPI network analyses

• Enrichr / WebGestalt: online tools for pathway enrichment analysis

2. Integration with PPI Network Analysis

Construction of protein–protein interaction (PPI) networks of acylated proteins enables the identification of hub nodes or functional modules within the network. When integrated with pathway information, this approach can:

• Reveal core regulatory modules

• Analyze coordinated modification networks (with crosstalk involving phosphorylation and ubiquitination)

• Identify potential drug targets or disease-associated mechanisms

It is recommended to use the STRING database for PPI network construction and to perform modular analyses using Cytoscape with plugins such as MCODE and CytoHubba.

Sources of Experimental Data and Quality Control Suggestions

At the early stage of a project, it is advisable to initiate downstream bioinformatics analyses to guide the rational design of upstream experiments. For example:

• High-resolution mass spectrometry platforms (such as Orbitrap Fusion Lumos) can be employed for acylation proteome profiling.

• Enrichment strategies may include anti-modification antibody-based enrichment (such as anti-acetyl-lysine) or chemical derivatization-based capture approaches.

• During data analysis, strict control of peptide identification confidence is required, with FDR < 1%, and site localization probability scoring should be performed (e.g., using the PTM Score in MaxQuant).

With the continued advancement of research in metabolic regulation and epigenetics, protein acylation has emerged as a critical hub linking metabolism, signal transduction, and epigenetic regulation, and is increasingly recognized as a focus of frontier research in the life sciences. Through systematic functional annotation and signaling pathway analysis, researchers can not only elucidate its biological functions but also provide a theoretical basis for disease mechanism studies and targeted drug development. If you are investigating the roles of acylation in specific diseases, metabolic pathways, or cellular states, MtoZ Biolabs offers customized experimental strategies and in-depth bioinformatics analysis support.

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

Related Services

Acylation Quantitative Proteomics Service