Compositional Analysis of Asymmetric and Symmetric
Dimethylated H3R2 Using Liquid
Chromatography–Tandem Mass Spectrometry-Based Targeted Proteomics
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Abstract
Protein
arginine methylation is one of the common post-translational
modifications in cellular processes. To date, two isomeric forms of
dimethylated arginine have been identified: asymmetric <i>N</i><sup>G</sup>,<i>N</i><sup>G</sup>-dimethylarginine (aDMA),
and symmetric <i>N</i><sup>G</sup>,<i>N</i>′<sup>G</sup>-dimethylarginine (sDMA). Evidence indicated that these isomers
can coexist and have different or even opposite functions, with aDMA
and sDMA forms of arginine 2 on histone H3 (i.e., H3R2me2a and H3R2me2s)
being an example. Thus, specific detection and quantification of each
isomeric form is important. Current methods are capable of predicting
and detecting thousands of methylarginine sites in proteins, whereas
differentiation and stoichiometric measurement of dimethylated protein
isomers are still challenging. Liquid chromatography coupled with
tandem mass spectrometry (LC–MS/MS)-based targeted proteomics
has emerged as a promising technique for site-specific quantification
of protein methylation using enzymatic peptides as surrogates of target
proteins. However, it should be pointed out that a routine targeted
proteomics strategy cannot easily distinguish sDMA- and aDMA-containing
surrogate peptides due to their common nature. The estimated amount
should be considered as the sum of both arginine dimethylated isomers.
In this study, compositional analysis based on a linear algebra algorithm
as an add-on to targeted proteomics was employed to quantify H3R2me2a
and H3R2me2s (i.e., surrogate peptides of AR(me2a)TK(me1/2)QT and
AR(me2s)TK(me1/2)QT). To achieve this simultaneous quantification,
a targeted proteomics assay was developed and validated for each isomer
first. With the slope and intercept of their calibration curves for
each multiple reaction monitoring (MRM) transition, linear algebraic
equations were derived. Using a series of mock mixtures consisting
of isomers in varying concentrations, the reliability of the method
was confirmed. Finally, the H3R2 dimethylation status was analyzed
in normal MCF-10A cells, parental drug-sensitive MCF-7/WT cancer cells,
and drug-resistant MCF-7/ADR cancer cells. Dimethylated H3R2 was also
monitored in MCF-7/WT cells with the treatment of doxorubicin (DOX)
for confirmation