Journal of Proteomics & Bioinformatics
Open Access

Implementation of Ubiquitinomics Methods in Protein Research Study

Jeroen A. Demmers^{* *}Correspondence: Jeroen A. Demmers, Department of Biotechnology, Erasmus University Medical Center, Rotterdam, The Netherlands, Email:

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Description

Since its discovery approximately 40 years ago, the Ubiquitin- Proteasome System (UPS) has been shown to control a wide range of biological functions, including protein homeostasis. When proteins are ubiquitylated, downstream effectors identify them and modify the protein abundance, activity, or localization. Interestingly, many diseases, including cancer and neurodegeneration, have downregulated ubiquitylation machinery. An innovative approach for assessing protein ubiquitylation objectively is Mass Spectrometry (MS)-based proteomics. To detect and quantify ubiquitylated proteins and map ubiquitylations at single amino acid resolution, MS-based proteomics has emerged as the most powerful and adaptable method. The groundbreaking discoveries of protein ubiquitylation and its function in proteasome degradation were made in the late 1970s and early 1980s and acknowledged with the Nobel Prize in Chemistry in 2004. Due to the complexity of the UPS, determining the activity and specificity of E3 ligases and DUBs, and discovering their substrates under physiological conditions, are challenging tasks. The preferred technique for determining the protein concentrations in cells and tissues is MS-based proteomics.

Protein-centric ubiquitinomics methods

Affinity tag chromatography, ubiquitin-specific antibodies, or ubiquitin-binding traps are some of the protein-centric techniques used to identify ubiquitin-modified proteins. The most typical method involves overexpressing His-tagged ubiquitin in cells, followed by immobilised metal affinity chromatography to separate ubiquitylated proteins through their tags under denaturing conditions. While avoiding copurification of non-covalently interacting binding partners, this protein-centric technique enables the measurement of hundreds of ubiquitylated proteins by MS. Cell lysis under denaturing conditions also helps in the quick inactivation of DUBs, maintaining protein ubiquitylation. On the other hand, ubiquitin overexpression might facilitate synthetic substrate ubiquitylation.

Peptide-centric ubiquitinomics methods

The measurement of so-called di-Gly residual peptides by MS is the gold standard technique for mapping ubiquitylation sites on target proteins globally. There are two glycine residues followed by an arginine at the C-terminus of ubiquitin, which is covalently joined to the lysine residues on target proteins. Thus, tryptic digestion of ubiquitylated proteins produces peptides with a missing cleavage lysine site and a distinctive di-Gly signature, increasing the peptide mass by 114.043 Da and allowing for MS separation from the corresponding unmodified parent peptides. The direct mapping of 110 ubiquitylation sites was made possible by expressing His-tagged ubiquitin and enriching ubiquitylated proteins in denaturing conditions, followed by thorough peptide fractionation before LC-MS analysis. However, the production of ubiquitylated peptides obtained through this method remained relatively low, due to the fact that proteins, rather than modified peptides, were enriched.

More recent mass spectrometer generations, together with ion mobility tools and new scan modes, have all contributed to the improvement of ubiquitinomics techniques. Tandem mass tag (TMT)-based ubiquitinomics was simplified and named UbiFast. In approximately 5 hours of MS time and with just 500 g of peptide input per sample, ion mobility-based MS and 10-plex TMT were utilized to quantify more than 10,000 ubiquitylation sites in tumour tissue across all samples. The key improvement of this method is the downsized TMT labelling, which is carried out while K-GG peptides are still linked to the antibody. It also uses less protein input. In order to improve throughput in ubiquitinomics, multiplexing through metabolic labelling such as neutron encoding (NeuCode) also provides an efficient alternative. Through the mapping of specific E3 ligase or DUB substrates, the analysis of cancer-specific ubiquitin signalling signatures, or studies on the mechanisms-of-action of UPStargeting drugs. The ubiquitin networks put together by Parkin were then characterized by MS-based ubiquitinomics, revealing K6-, K11-, and K63-linked polymers. Numerous facets of the cell's response to DNA damages are known to be controlled by ubiquitylation.The analysis of DNA damage signalling has benefited greatly from the use of MS-based ubiquitinomics over time. After subjecting cells to UV light, Researchers used MSbased ubiquitinome profiling for the first time, identifying multiple ubiquitylation events that not had been identified earlier. The UPS is crucial to almost every cellular action, and disorders. It have been associated with m any type of disorders. Such neosubstrates have indeed been successfully detected as being ubiquitylated after medication treatment using K-GGbased ubiquitinomics. The use of MS in ubiquitinomics research will increase in the future, making it possible to precisely and spatiotemporally examine ubiquitin signalling.