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Proteome-wide enrichment of proteins modified by lysine methylation

Nature Protocols volume 9, pages 37–50 (2014)Cite this article

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Abstract

We present a protocol for using the triple malignant brain tumor domains of L3MBTL1 (3xMBT), 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 3xMBT, 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 3xMBT 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.

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Figure 1: Overview and experimental design.
Figure 2: Example of silver-stained gels showing protein pull-down by 3xMBT from protein extract of human cells.
Figure 3: Application of methyl-lysine enrichment to Rkm1 substrate identification.

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Acknowledgements

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.

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Author notes

  1. Erin M Green

    Present address: Present address: Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland, USA.,

  2. Scott M Carlson and Kaitlyn E Moore: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Stanford University, Stanford, California, USA

    Scott M Carlson, Kaitlyn E Moore, Erin M Green, Glòria Mas Martín & Or Gozani

  2. Department of Chemical and Systems Biology, School of Medicine, Stanford University, Stanford, California, USA

    Kaitlyn E Moore

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  1. Scott M Carlson
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Contributions

S.M.C. and K.E.M. developed the 3xMBT pull-down protocol. S.M.C. optimized the protocol for mass spectrometry. E.M.G. conducted the Rkm1 experiment. G.M.M. generated SILAC yeast strains and adapted the protocol to yeast. S.M.C. and K.E.M. wrote the protocol. O.G. supervised the work.

Corresponding authors

Correspondence to Scott M Carlson or Or Gozani.

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Competing interests

O.G. is a cofounder of EpiCypher, Inc. Stanford University has submitted a provisional US patent describing the use of the 3xMBT domain as an affinity reagent.

Supplementary information

Supplementary Table 1

Protein identification and SILAC data from 3xMBT pull-down of lysate from yeast Δrkm1 and wild-type cells. (XLSX 17 kb)

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Carlson, S., Moore, K., Green, E. et al. Proteome-wide enrichment of proteins modified by lysine methylation. Nat Protoc 9, 37–50 (2014). https://doi.org/10.1038/nprot.2013.164

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