Pupylation Proteomics Service

Pupylation is a prokaryote-specific post-translational modification (PTM) predominantly found in Actinobacteria such as Mycobacterium tuberculosis. In this modification, a small protein called Pup (prokaryotic ubiquitin-like protein) is covalently attached to the lysine residues of target proteins, leading to functional regulation or degradation. While functionally similar to ubiquitination in eukaryotes, Pupylation differs significantly in its structural components and enzymatic mechanisms.

 



Striebel, F. et al. Biochim Biophys Acta. 2014.

Figure 1. Reaction Scheme of Ubiquitination in Eukaryotes and the Bacterial Pupylation Pathway

 

Recent studies have linked Pupylation to protein homeostasis regulation, bacterial stress adaptation, virulence modulation, and antibiotic resistance development. Particularly, Pupylation is emerging as a promising avenue for anti-tuberculosis drug target research. Since this system is absent in human cells, enzymes such as PafA (ligase) and Dop (deamidase) offer attractive selectivity for drug development.

 

Leveraging a high-sensitivity mass spectrometry platform and deep expertise in proteomics, MtoZ Biolabs has established a comprehensive analytical workflow to provide end-to-end Pupylation Proteomics Service, supporting the systematic study of Pupylation's biological significance.

 

Analysis Workflow

MtoZ Biolabs offers a standardized yet flexible Pupylation proteomics workflow, including:

1. Sample Preparation and Assessment

Clients submit total protein extracts from bacterial samples. We perform concentration measurement, SDS-PAGE evaluation, and initial quality control to ensure analytical reliability.

 

2. Enrichment of Pupylated Proteins

Depending on project needs, we employ anti-Pup immunoprecipitation or tag-based affinity purification methods to enrich low-abundance modified proteins.

 

3. Proteolytic Digestion and Peptide Cleanup

Enriched samples are digested under optimized enzymatic conditions and purified using approaches such as StageTips for desalting and concentration.

 

4. LC-MS/MS Analysis

High-resolution mass spectrometry platforms (e.g., Orbitrap, Q-Exactive series) are used to detect modified peptides and acquire comprehensive MS and MS/MS data.

 

5. Data Analysis and Annotation

Software such as Proteome Discoverer and MaxQuant is used for database search, Pup site identification, quantification, and functional enrichment analysis.

 

6. Report Delivery and Technical Support

Clients receive a structured report, including Pupylation site lists, peptide spectra, functional annotations, and visual summaries. Follow-up interpretation and consultation are available.

 

Service Advantages

1. Pupylation-Focused Expertise: Tailored for bacteria-specific modification systems, optimized for Actinobacteria targets.

 

2. High-Sensitivity MS Platform: Enables detection of low-abundance and dynamically regulated Pupylated proteins.

 

3. Modular and Customizable Workflow: Adaptable enrichment strategies and database configurations according to research needs.

 

4. In-Depth Bioinformatics Support: Integration of site-level data with pathway enrichment to build functional regulatory models.

 

5. Standardized Deliverables: Clear, publication-ready reports facilitate downstream analysis and research translation.

 

Applications

MtoZ Biolabs' Pupylation proteomics service is applicable to a wide range of research areas:

1. Bacterial Protein Degradation Pathways

Dissecting the role of Pupylation in maintaining protein stability and cellular proteostasis.

 

2. Anti-Tuberculosis Drug Target Discovery

Structural and functional profiling of Pup system enzymes such as PafA and Dop to guide inhibitor development.

 

3. Stress Response and Virulence Factor Regulation

Comparative analysis of Pupylation profiles under different environmental or host-infection conditions.

 

4. Mutant Strain Functional Characterization

Comparing Pupylation differences between wild-type and mutant strains to validate key regulatory proteins.

 

5. Structure–Function Correlation Studies

Providing site-specific modification data to complement structural biology methods like Cryo-EM and X-ray crystallography.

 

FAQ

Q1: What types of samples are suitable for Pupylation analysis?

A1: The service is mainly applicable to bacterial samples, especially Actinobacteria. Samples should be collected during metabolically active phases to ensure sufficient modification levels.

 

Q2: Do you provide Pup enrichment reagents?

A2: We can recommend commercially available antibodies or offer guidance on tag design. Reagents can also be sourced upon request.

 

Q3: Can unknown or novel Pupylation sites be detected?

A3: Yes. We support open modification searches and tag-based detection to identify both known and novel Pup sites.

 

Q4: Can non-model bacterial strains be analyzed?

A4: Custom databases are supported, making the service compatible with non-model organisms, environmental isolates, and mutant strains.

 

Q5: What is the turnaround time for this service?

A5: Standard projects are typically completed within 4–6 weeks. For large-scale or customized database projects, timelines may be extended accordingly.

 

To learn more about MtoZ Biolabs’ Pupylation Proteomics Service or to request a project consultation, please contact our technical support team. We are committed to delivering reliable, professional solutions to advance your research in microbial regulation and drug development.