Updated project metadata. We have presented a protocol that utilizes the triple malignant brain tumor domains of L3MBTL1 (3xMBT) to enrich proteins modified with mono- and di-methylated lysine from cell lysate. 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) is then used to identify proteins that are specifically enriched by 3xMBT pull-down. Addition of a third isotopic label allows comparison of protein methylation between biological conditions. As an example yeast cells were prepared in SILAC media under three conditions. In two independent experiments either light or heavy cells are Rkm1 knock-out, while the other two are wild-type. 3xMBT was used to enrich methylated proteins from the light/heavy cells, with 3xMBT_D355N as a negative control using the medium cell lysate. Proteins were separated into three fractions by SDS-PAGE and analyzed by LC-MS/MS using an Orbitrap Velos. Data was analyzed with MaxQuant version 1.3.0.5 using default parameters except that mono- and di-methylation of lysine were included as variable modifications, the modification-specific false-discovery was set to 10%, and minimum peptide count for quantitative data set to 1. The amount of ribosomal protein Rpl23, a known substrate of Rkm1, is greatly decreased in 3xMBT pull-down from Rkm1 knock-out cells relative to wild-type. The level of heat shock protein SSA4 is also greatly reduced, suggesting that it may also be methylated by Rkm1.