Peptide Level Immunoaffinity Enrichment Enhances Ubiquitination Site Identification on Individual Proteins
Molecular & Cellular Proteomics, ISSN: 1535-9476, Vol: 13, Issue: 1, Page: 145-156
2014
- 25Citations
- 76Captures
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Example: if you select the 1-year option for an article published in 2019 and a metric category shows 90%, that means that the article or review is performing better than 90% of the other articles/reviews published in that journal in 2019. If you select the 3-year option for the same article published in 2019 and the metric category shows 90%, that means that the article or review is performing better than 90% of the other articles/reviews published in that journal in 2019, 2018 and 2017.
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Metrics Details
- Citations25
- Citation Indexes25
- 25
- CrossRef23
- Captures76
- Readers76
- 76
Article Description
Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.
Bibliographic Details
http://www.sciencedirect.com/science/article/pii/S1535947620342663; http://dx.doi.org/10.1074/mcp.m113.031062; http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=84891801891&origin=inward; http://www.ncbi.nlm.nih.gov/pubmed/24142993; http://www.mcponline.org/lookup/doi/10.1074/mcp.M113.031062; https://syndication.highwire.org/content/doi/10.1074/mcp.M113.031062; https://linkinghub.elsevier.com/retrieve/pii/S1535947620342663; https://dx.doi.org/10.1074/mcp.m113.031062; https://www.mcponline.org/content/13/1/145; https://www.mcponline.org/content/13/1/145.abstract; https://www.mcponline.org/content/mcprot/13/1/145.full.pdf; http://www.mcponline.org/content/13/1/145; http://www.mcponline.org/content/13/1/145.abstract; http://www.mcponline.org/content/13/1/145.full.pdf; http://europepmc.org/abstract/med/24142993; http://europepmc.org/articles/PMC3879610; http://www.mcponline.org/cgi/doi/10.1074/mcp.M113.031062
American Society for Biochemistry & Molecular Biology (ASBMB)
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