dc.contributor.author | Carlson, Scott M | |
dc.contributor.author | Moore, Kaitlyn E | |
dc.contributor.author | Green, Erin | |
dc.contributor.author | Martín, Glòria Mas | |
dc.contributor.author | Gozani, Or | |
dcterms.creator | https://orcid.org/0000-0003-3923-6726 | en_US |
dc.date.accessioned | 2023-01-25T20:47:54Z | |
dc.date.available | 2023-01-25T20:47:54Z | |
dc.date.issued | 2013-12-05 | |
dc.description.abstract | We present a protocol for using the triple malignant brain tumor domains of L3MBTL1 (3×MBT), which bind to mono- and di-methylated lysine with minimal sequence specificity, in order to enrich for such methylated lysine from cell lysates. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3×MBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) are then used to identify proteins that are specifically enriched by 3×MBT pull-down. The addition of a third isotopic label allows the comparison of protein lysine methylation between different biological conditions. Unlike most approaches, our strategy does not require a prior hypothesis of candidate methylated proteins, and it recognizes a wider range of methylated proteins than any available method using antibodies. Cells are prepared by growing in isotopic labeling medium for about 7 d; the process of enriching methylated proteins takes 3 d and analysis by LC-MS/MS takes another 1–2 d. | en_US |
dc.description.sponsorship | This work was supported in part by a grant from the US National Institutes of Health (NIH) to O.G. (R01 GM079641). S.M.C. was supported by an American Cancer Society Illinois Division Postdoctoral Fellowship award 123711-PF-13-093-01-TBE. K.E.M. was supported by a Hubert Shaw and Sandra Lui Stanford Graduate Fellowship. This material is based on work supported by the National Science Foundation Graduate Research Fellowship under grant no. DGE-1147470 to K.E.M. G.M.M. was supported by the Generalitat de Catalunya Beatriu de Pinos award BP-A 2010. O.G. is a recipient of an Ellison Senior Scholar in Aging Award. We thank the PRIDE team for assistance in making mass spectrometry data available. | en_US |
dc.description.uri | https://www.nature.com/articles/nprot.2013.164 | en_US |
dc.format.extent | 27 pages | en_US |
dc.genre | journal articles | en_US |
dc.genre | postprints | en_US |
dc.identifier | doi:10.13016/m2ypqk-pdpi | |
dc.identifier.citation | Carlson, Scott M et al. “Proteome-wide enrichment of proteins modified by lysine methylation.” Nature protocols vol. 9,1 (2014): 37-50. doi:10.1038/nprot.2013.164 | en_US |
dc.identifier.uri | https://doi.org/10.1038/nprot.2013.164 | |
dc.identifier.uri | http://hdl.handle.net/11603/26714 | |
dc.language.iso | en_US | en_US |
dc.publisher | Nature | en_US |
dc.relation.isAvailableAt | The University of Maryland, Baltimore County (UMBC) | |
dc.relation.ispartof | UMBC Biological Sciences Department Collection | |
dc.rights | This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1038/nprot.2013.164 | en_US |
dc.title | Proteome-wide enrichment of proteins modified by lysine methylation | en_US |
dc.type | Text | en_US |