Quantitative Analysis of Nonhistone Lysine Methylation Sites and Lysine Demethylases in Breast Cancer Cell Lines

Growing evidence
shows that lysine methylation is a widespread
protein post-translational modification (PTM) that regulates protein
function on histone and nonhistone proteins. Numerous studies have
demonstrated that the dysregulation of lysine methylation mediators
contributes to cancer growth and chemotherapeutic resistance. While
changes in histone methylation are well-documented with extensive
analytical techniques available, there is a lack of high-throughput
methods to reproducibly quantify changes in the abundances of the
mediators of lysine methylation and nonhistone lysine methylation
(Kme) simultaneously across multiple samples. Recent studies by our
group and others have demonstrated that antibody enrichment is not
required to detect lysine methylation, prompting us to investigate
the use of tandem mass tag (TMT) labeling for global Kme quantification
without antibody enrichment in four different breast cancer cell lines
(MCF-7, MDA-MB-231, HCC1806, and MCF10A). To improve the quantification
of KDMs, we incorporated a lysine demethylase (KDM) isobaric trigger
channel, which enabled 96% of all KDMs to be quantified while simultaneously
quantifying 326 Kme sites. Overall, 142 differentially abundant Kme
sites and eight differentially abundant KDMs were identified among
the four cell lines, revealing cell line-specific patterning.