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Ubiquitinated Proteomics Analysis

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What Is Protein Ubiquitination

Protein ubiquitination is a prevalent and essential post-translational modification (PTM) in eukaryotes. It is the process that ubiquitin (Ub) links to lysine (K) residues and N-terminal methionine (M) sites on proteins through a cascade mechanism catalyzed by enzymes (E1, E2, E3). Ultimately, the 26S proteasome specifically targets and degrades the ubiquitinated proteins. Reversibly, under the catalysis of deubiquitinases (DUB), Ub can be dissociated from substrate. Additionally, Ub containing seven K and one M at N-terminal, Ub covalently attached to proteins can still be further modified by other Ubs to form poly-Ubs (figure 1). According to the number of Ub on protein, it can be divided into mono-ubiquitination and poly-ubiquitination, and poly-ubiquitination can be divided into linear poly-ubiquitination chain and non-linear poly-ubiquitination chain. In a word, a protein may have multiple ubiquitination sites, and each site may be linked to more than one ubiquitination molecule and contains unique functions.

The location and amount of protein ubiquitination play a crucial role in determining its functions. It plays a pivotal regulatory role in all biological processes, especially in regulating protein quality control, encompassing the degradation of misfolded, dysfunctional, or otherwise aberrant and unnecessary proteins. When there is aberrant expression of Ub or functional mutations in E1, E2, or E3 enzymes, it can lead to abnormal protein degradation and accumulation, potentially contributing to the development of various diseases. Therefore, a systematic study on ubiquitination helps us gain a profound understanding of the pathogenicity molecular mechanism and develop novel strategies to combat diseases.

Figure 1. The cycle of Ub signaling and Ub proteoformsFigure 1. The cycle of Ub signaling and Ub proteoforms[1].
(A) The UPS. (B) The various forms of polyUb-chain.

Table 1. Individual ubiquitin linkage types and their associated biological roles [2].

Types of ubiquitin chainsFunctions
K61) Mitophagy; 2) DNA damage response
K111) Cell cycle and mitosis regulation; 2) Membrane trafficking
3) Mitophagy
K271) DNA damage response; 2) Host response to microbial DNA
3) T-cell development; 4) Proteasomal signaling
K291) Proteasomal signaling
K331) Regulation of TCR response
K481) Proteasomal Degradation
K631) Regulation of NF-κB signaling; 2) Transcriptional activation
3) DNA damage response; 4) Lysosomal targeting
a. Autophagy b. Receptor internalization;
5) Protein-protein interactions
M11) Regulation of NF-κB signaling; 2) Protein kinase activation
3) Protein-protein interactions

The principle for identification of ubiquitination sites based on LC-MS/MS

The molecular mass of protein peptides or amino acids undergoing ubiquitination will correspondingly increase. For a known protein or peptide, its molecular mass is already established. Trypsin digestion on ubiquitinated protein leaves a di-glycine remnant and LC-MS/MS can detect this change in molecular mass addition and determine the specific amino acid site where ubiquitination modification occurs.

Our Ubiquitinated Proteomics Service

At Creative Proteomic, the comprehensive protein ubiquitination analysis services including protein extraction, digestion, ubiquitinated peptide enrichment (highly specific antibody), LC-MS/MS analysis, data and bioinformatics analysis, report preparation. For large-scale, high-throughput, and deep coverage in identification, we can also combine fractionation methods at protein or peptide level. Additionally, technologies such as TMT/iTRAQ could also help to achieve precise quantification among different samples.

Creative Proteomics is a reliable choice for your projects. Because Creative Proteomics boasts a team of highly skilled and knowledgeable PhD researchers specializing in PTMs proteomics, with over ten years of experience in ubiquitinated-proteomics analysis. We can quickly and efficiently catch and analyze the dynamic changes of ubiquitinated proteins to meet your needs, assisting in biomarker identification and drug target screening accurately.

Figure 2. MS-based approaches for identifying ubiquitination sitesFigure 2. MS-based approaches for identifying ubiquitination sites[3].

Technological superiority

  1. Professional detection and analysis capability: Experienced PTM technical team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensure reliable and accurate data.
  2. Reproducible: Obtain consistent and reproducible inter- and intra- assay results for data analysis.
  3. High specificity, high-throughput: Identify and quantify up to thousands of proteins at once and deeper coverage of ubiquitination peptides and sites.
  4. High resolution and sensitivity: Q-Exactive, Q-Exactive HF, Orbitrap Fusion™ Tribrid™, etc.
  5. Short period, high quality, and the results deliver quickly.

Samples Requirement

We can accept a variety of samples, including but not limited to:

Tissue: animal tissue > 100 mg;

fresh plant > 500 mg;

Cell: suspension cell > 5 x 107;

adherent cell > 5 x 107;

microorganism > 100 mg or 5 x 107 cells;

Body fluid: serum/plasma > 1 mL;

Protein: total protein >5 mg and concentration >1 μg/μL.

Note: In order to ensure the test results, please inform the buffer components if you give us proteins, whether it contains thiourea, SDS, or strong ion salts. In addition, the sample should not contain components such as nucleic acids, lipids, and polysaccharides, which will affect the separation effect.

Results Delivery

  1. Detailed report, including experiment procedures, parameters, etc.
  2. Raw data and data analysis results.

How to place an order:

Creative Proteomics offers a diverse range of cutting-edge technologies for ubiquitin research, facilitating precise quantification of protein levels and accurate identification of ubiquitination sites. Please feel free to contact us to discuss your specific needs. Our customer service representatives are available 24 hours a day, from Monday to Sunday.


  1. Sun M, Zhang X. Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture. Cell & Bioscience. 2022 Aug 12;12(1):126.
  2. Mooney EC, Sahingur SE. The Ubiquitin System and A20: Implications in Health and Disease. Journal of Dental Research. 2021 Jan;100(1):10-20.
  3. Ordureau A, Münch C, Harper JW. Quantifying ubiquitin signaling. Molecular Cell. 2015 May 21;58(4):660-76.

Rewiring of the ubiquitinated proteome determines ageing in C. elegans

Journal: Nature

Published: 2021

Main Technology: Label-free quantitative ubiquitinated proteomics


Ubiquitination is one of the most common protein degradation pathways in eukaryotes. By labeling proteins with ubiquitination, they are recognized and degraded by the 26S proteasome in cell (this process can be reversed by the deubiquitinating enzymes). The attachment of the small protein ubiquitin to lysine residues of specific targets is a central pathway by which cellular decisions are made, but the effect of ubiquitination in ageing remains unclear.

The identification results of Ub-proteome

We compared the ubiquitin (Ub)-modified proteome of worms at the first day of adulthood with young (day 5), mid-age (day 10) and aged adults (day 15). With high reproducibility between biological replicates, our assay identified ubiquitination sites for 3,373 peptides that correspond to 1,485 distinct proteins. With more analysis, given that C. elegans undergoes a widespread proteome remodelling during ageing, differences in ubiquitination levels could not be simply ascribed to a similar change in the protein amounts.

Research results

  1. Remodeling of ubiquitinated proteomes occurs during aging.
  2. Deubiquitinating enzyme inhibited protein ubiquitination occurs with age.
  3. Degradation of proteasome targets is hindered with age, Proteasome targets determine lifespan.
  4. Illustrated two of proteasomal targets biological function, IFB-2 intermediate filament and the EPS-8 modulator of RAC signalling.

Ubiquitinated Proteomics AnalysisFigure 1. Rewiring of the Ub-proteome with age.

* For Research Use Only. Not for use in diagnostic procedures.
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