Isolation and Mass Spectrometry Identification of K48 and K63 Ubiquitin Proteome Using Chain-Specific Nanobodies
Protein ubiquitylation is an essential mechanism regulating almost all cellular functions in eukaryotes. The understanding of the role of distinct ubiquitin chains in different cellular processes is essential to identify biomarkers for disease diagnosis and prognosis but also to open new therapeutic...
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| creator | Gonzalez-Santamarta, Maria Ceccato, Laurie Carvalho, Ana Sofia Rain, Jean-Christophe Matthiesen, Rune Rodriguez, Manuel S. |
| description | Protein ubiquitylation is an essential mechanism regulating almost all cellular functions in eukaryotes. The understanding of the role of distinct ubiquitin chains in different cellular processes is essential to identify biomarkers for disease diagnosis and prognosis but also to open new therapeutic possibilities. The high complexity of ubiquitin chains complicates this analysis, and multiple strategies have been developed over the last decades. Here, we report a protocol for the isolation and identification of K48 and K63 ubiquitin chains using chain-specific nanobodies associated to mass spectrometry. Different steps were optimized to increase the purification yield and reduce the binding on nonspecific proteins. The resulting protocol allows the enrichment of ubiquitin chain-specific targets from mammalian cells. |
| doi_str_mv | 10.1007/978-1-0716-2859-1_9 |
| format | Book Chapter |
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| fulltext | fulltext |
| identifier | ISSN: 1064-3745 |
| ispartof | Methods in molecular biology (Clifton, N.J.), 2023, Vol.2602, p.125-136 |
| issn | 1064-3745 1940-6029 |
| language | eng |
| recordid | cdi_hal_primary_oai_HAL_hal_04269347v1 |
| source | Springer Books |
| subjects | Cellular Biology Isolation Life Sciences Mass spectrometry Nanobodies Posttranslational modifications Proteome Ubiquitin |
| title | Isolation and Mass Spectrometry Identification of K48 and K63 Ubiquitin Proteome Using Chain-Specific Nanobodies |
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