Characterization of Protein SUMOylation

What is SUMOylation?

The Small Ubiquitin-related Modifier (SUMO) is a family of small proteins, each composed of approximately 100 amino acids (AAs). Notably, the three-dimensional structure reveals a striking resemblance between SUMO and ubiquitin, despite sharing less than 20% identity in their AA sequences and exhibiting significant differences in overall surface-charge distribution. The human genome encodes five SUMO proteins, namely SUMO1 through SUMO5. The similarity between SUMO1 and SUMO2, as well as SUMO3, is approximately 50%, while the similarity between SUMO1 and SUMO5 is approximately 88%. On the other hand, SUMO2 and SUMO3 exhibits a high degree of similarity (97%, differing only in three N-terminal residues) and is often referred to as SUMO-2/3.

Table 1. The AAs sequences of SUMO1-5 in human.

Similar to ubiquitination mechanism, SUMOylation refers to the process of specifically covalently linking the C-terminus residue of mature SUMO proteins to the ε-amino group of lysine (K) residues on acceptor substrate proteins. Besides, the process of SUMOylation involves a cascade of enzymatic reactions facilitated by the SUMO activating enzyme, conjugating enzyme and ligase (E1, E2, E3). However, different from ubiquitination, SUMO protein needs to undergo a mature process before it can proceed to SUMOylation, that is, the SUMO protein precursor is hydrolyzed to their mature forms by sentrin-specific peptidases (SENPs) to release a carboxy–terminal Glycine–Glycine (GG) motif. Reversibly, deSUMOylation is mediated by the catalytic activity of SENPs and free SUMO can be recycled again for another round of protein conjugation.

Figure. The mechanisms of the SUMO pathway ^[1].

The SUMOylation is a ubiquitous, highly dynamic and reversible type of protein post-translational modification (PTM) that has been identified as being widely involved in controlling cellular physiological and pathological processes, including genome maintenance, gene expression, protein stability, protein subcellular localization, and signal transduction. As a competitor of ubiquitination, SUMOylation has become one of the research hotspots in recent years. Many reports shows that it is abnormally activated in the occurrence and development of various tumors, such as cardiovascular diseases, cancer, neurodegenerative diseases, and immune diseases. However, the functional relevance of SUMOylation remains to be thoroughly investigated and validated.

Our Characterization of Protein SUMOylation Service.

The presence of threonine (T) preceding the GG at the C terminus of SUMO1-4 enables specific enzymatic digestion of the SUMOylation protein using the Wild Type Alpha-Lytic Protease (WaLP), resulting in a peptide fragment contains a K-ε-GG residue. The WaLP protease is a serine (S) endopeptidase that cleaves at the carboxyl terminal side of T, S, alanine (A), and valine (V) residues with high specificity. The fundamental workflow is as follows:

1. The experiment designed (clear intention);
2. Choice of MS (Mass Spectrometry) instrument and analysis method;
3. Methods optimization;
4. Sample preparation—protein extraction and determination of concentration;
5. Utilize WaLP protease for proteolytic digestion;
6. Enrichment of SUMOylated peptides by K-ε-GG antibody;
7. Peptides fractionations for in-depth identification by C₁₈ StageTips or reversed phase high performance liquid chromatography (optional);
8. LC-MS/MS analysis;
9. Bioinformation analysis for full SUMOylation proteins, SUMOylated peptides and SUMOylated sites annotation.
10. Deliver high-quality technical reports.

Figure 2. General workflow elements for characterization of SUMOylation in biological samples.

Furthermore, we excel in providing SUMOylation quantitative proteomics service as well, particularly in SILAC, iTRAQ/TMT, label-free, DIA and target proteomics (SRM/PRM). Creative Proteomics also provides an effective platform for conducting targeted investigations on the SUMOylated sites of SUMO1 to SUMO4  - conjugated substrate proteins. These technologies facilitate the full analysis and interpretation of SUMOylation.

The Advantages of the SUMOylation characterized service in Creative Proteomics.

1. Highly accurate and reliable quantification results: The utilization of advanced MS technology, well-established protein quantification methods, along with integration of SUMOylation protein or peptide enrichment strategies, ensures precise and high-confidence identification and quantification results of SUMOylation.
2. Various sample types: Possessed extensive experience in handling diverse sample, such as archaea, prokaryotes, cells, tissues, and bodily fluids, etc.
3. Professional detection and analysis capability: Experienced SUMOylation identification and quantification technical team, strict quality control system, together with ultra-high resolution detection system and proficient data pre-processing and analysis capability, ensures the delivery of reliable and accurate data.
4. Reproducible: Obtain consistent and reproducible inter- and intra- assay results for data analysis.
5. High throughput and sensitivity: Q-Exactive, Q-Exactive HF, Orbitrap Fusion™ Tribrid™, etc.
6. The results are delivered in a short-term and of high quality.

Samples Requirement

Tissue: animal tissue > 50 mg;

fresh plant > 100 mg;

Cell: suspension cell > 5 x 10⁷;

adherent cell > 5 x 10⁷;

microorganism > 50 mg or 5 x 10⁷ cells;

Body fluid: serum/plasma > 500 μL;

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

Creative Proteomics also provide the following services in PTMs Analysis:

1. Predict PTM modification sites using bioinformatics;
2. Further validate the identified PTM-proteins or PTM-sites;
3. Functional annotation and enrichment analysis;
4. Clustering analysis;
5. Network analysis;
6. Statistical analysis.

How to place an order

With over 10 years experimental experience, Creative Proteomics have the expertise to provide a tailored and scientific experimental scheme that meets your specific requirements for conducting SUMOylation modification analysis. Please feel free to contact us by email to discuss your specific needs. Our customer service representatives are available 24 hours a day, from Monday to Sunday.

Reference

1. Fan Y, Li X, Zhang L, et al. SUMOylation in Viral Replication and Antiviral Defense. Advanced Science. 2022 Mar;9(7):e2104126.

Site-specific identification and quantitation of endogenous SUMO modifications under native conditions

Journal: Nature Communications

Published: 2017

Main Technology: shotgun proteomics, Ubiquitinated-proteomics, SILAC-based quantitative proteomics.

Abstract

Small ubiquitin-like modifier (SUMO) modification regulates numerous cellular processes. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. Proteome-wide detection of SUMOylation sites on target proteins typically requires ectopic expression of mutant SUMOs with introduced tryptic sites. Here, we report a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP. WaLP digestion of SUMOylated proteins generates peptides containing SUMO-remnant diglycyl-lysine (KGG) at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, we identified 1209 unique endogenous SUMO modification sites. We also demonstrate the impact of proteasome inhibition on ubiquitin and SUMO-modified proteomes using parallel quantitation of ubiquitylated and SUMOylated peptides. This methodological advancement enables determination of endogenous SUMOylated proteins under completely native conditions.

Figure 1. A strategy for mapping endogenous SUMO-modification sites. a C-terminal sequence alignment of processed human SUMO1-4 and ubiquitin. The proximal amino acid to the diGlycine C-terminal residues is indicated in red. b Schematic depicting the Ub site mapping strategy. Proteins modified by ubiquitin are digested with trypsin leaving a diGlycine attached to the ε-amine of the lysine where ubiquitin was attached. An antibody specific for KGG-peptides is used to enrich peptides from ubiquitylated sites that are then identified by mass spectrometry. c The same as b but WaLP digestion is used to generate the KGG-peptides from SUMO attachment sites