Summary Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or di-methylated lysine. The domain is broadly specific for methylated lysine (“pan-specific”) and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of novel methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferse GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.

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.

The dynamic modification of histone proteins by lysine methylation has emerged over the last decade as a key regulator of chromatin functions. In contrast, our understanding of the biological roles for lysine methylation of non-histone proteins has progressed more slowly. Though recently it has attracted less attention, ε-methyl-lysine in non-histone proteins was first observed over 50 years ago. In that time, it has become clear that, like the case for histones, non-histone methylation represents a key and common signaling process within the cell. Recent work suggests that non-histone methylation occurs on hundreds of proteins found in both the nucleus and the cytoplasm, and with important biomedical implications. Technological advances that allow us to identify lysine methylation on a proteomic scale are opening new avenues in the non-histone methylation field, which is poised for dramatic growth. Here, we review historical and recent findings in non-histone lysine methylation signaling, highlight new methods that are expanding opportunities in the field, and discuss outstanding questions and future challenges about the role of this fundamental post-translational modification (PTM).

Background: SETMAR is a lysine methyltransferase (KMT) that contributes to DNA repair, but its biochemical function is not well understood. Results: A novel proteomic strategy identifies splicing factor snRNP70 as a SETMAR substrate. Conclusion: SETMAR is the first KMT identified to target splicing factors. Significance: Proteomics can be harnessed to discover methyltransferase substrates. Lysine methylation may be a new mode of regulation for mRNA splicing.

Introduction: Acute Myeloid Leukemia (AML) is clinically and biologically heterogeneous, requiring the detection of multiple mutations for characterization. For instance, FLT3-ITDs and CEBPA mutations represent important markers in AML, however they are difficult to detect by NGS due to the highly variable nature of ITDs, the high GC content of CEBPA, and the difficulty in mapping repeated sequences to a wild-type reference. Tracking low frequency mutations is also of growing importance. The ability to accurately detect variants at low allele fractions (AFs) using a single test can be used to assess treatment efficacy and potential relapse. Methods: We developed Archer® VariantPlex® myeloid assays based on Anchored Multiplex PCR (AMP™) to detect important mutations in myeloid malignancies. AMP is a target enrichment strategy that uses molecular-barcoded adapters and gene-specific primers for amplification, permitting open-ended capture of DNA fragments from a single end. This approach enables flexible and strand-specific primer design to provide better coverage of ITD-containing and GC-rich regions. We also developed a method to assess SNV sensitivity taking into account both unique coverage depth and noise for single base substitutions. This strategy enables utilization of position-specific detection thresholds and maximizes sensitivity and specificity. We tested this approach using the VariantPlex® Core Myeloid panel, by titrating reference inputs into background normal samples to examine detection of low AF variants. Results: Our assay enables calling of a 30bp FLT3-ITD down to sub-0.05% allele frequencies. Using optimized low AF conditions improves coverage depth, consistency of low AF FLT3-ITD detection, and sensitivity (98.5% of bases are powered to call a true variant at an allele frequency of 3.0% with 1M reads and 200ng of input). We show >1000X unique molecule coverage across the coding region of CEBPA and use this challenging region to visualize the minimum detectable AF (MDAF) at which a variant has a >95% probability of being detected above the noise (95MDAF). Finally, we show consistent single nucleotide variant (SNV), insertion and deletion (indel), and ITD calling at sub-0.5% allele frequencies, and demonstrate the utility of reporting variant-specific MDAFs and normal dataset P-values when analyzing low AF variants. Conclusion: AMP provides NGS-based detection of complex mutation types that are relevant in AML. We demonstrate robust calling of FLT3-ITDs and other variants at low AFs. We also demonstrate full coverage of CEBPA with high depth and low noise such that the 95MDAF predicts confident variant calling at low AFs. This approach is accurate and scalable, enabling simultaneous detection of multiple mutation types across multiple target genes in a single assay. Citation Format: Verity Johnson, Kaitlyn E. Moore, Laura M. Griffin, Aaron Berlin, Abel Licon, Ryan Walters. Accurate detection of low AF variants relevant to AML by Anchored Multiplex PCR and next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1699.