Quantitative Analysis and Discovery of Lysine and Arginine Modifications

Abstract

Post-translational modifications (PTMs) affect protein function, localization, and stability, yet very little is known about the ratios of these modifications. Here, we describe a novel method to quantitate and assess the relative stoichiometry of Lys and Arg modifications (QuARKMod) in complex biological settings. We demonstrate the versatility of this platform in monitoring recombinant protein modification of peptide substrates, PTMs of individual histones, and the relative abundance of these PTMs as a function of subcellular location. Lastly, we describe a product ion scanning technique that offers the potential to discover unexpected and possibly novel Lys and Arg modifications. In summary, this approach yields accurate quantitation and discovery of protein PTMs in complex biological systems without the requirement of high mass accuracy instrumentation.

Figure 1. A generalized scheme of the QuARKMod platform

QuARKMod has been validated for use with a broad range of cell and tissue samples. Following protein digestion, the absolute concentrations of PTMs can be determined in a given sample. In addition, using data-dependent scanning, we outline a method for the discovery of novel or low-abundance Lys and Arg PTMs in the same biological samples.

Figure 2. The application of QuARKMod for the study of in vitro enzyme activity

QuARKMod was used to investigate the overall methylation status of a synthetic peptide (2 nmol) containing a single me₃Lys following incubation with a recombinant Lys demethylase, JMJD2A. A quantitative yield was observed in total methylated Lys following incubation with JMJD2A with me₂Lys being the predominant PTM. The peptide sequence was as follows: ARTKQTARKme₃STGGKA.

Figure 3. Quantitation of Lys and Arg PTMs on isolated histones

(a) To measure histone-specific changes, histones were purified using an offline HPLC method, resulting in significant separation of each core histone. (b) Purified histones collected from HEK293 cells demonstrate varying degrees of Lys and Arg PTMs.

Figure 4. QuARKMod reveals distinct patterns of PTMs in subcellular fractions

Measurement of Lys and Arg PTMs in subcellular isolations from murine liver reveals that nuclear preparations contain the highest levels of modifications. Mitochondrial fractions contain high levels of acLys, me₃Lys, and ADMA.

Figure 5. Product ion scanning can be utilized for the discovery of novel Lys and Arg PTMs

(a) Profile of analytes with a product ion of 84.0 m/z (Lys) collected from chromatin isolated from KLA (red) or vehicle control (blue)-treated RAW264.7 macrophages. (b) XIC of 217.3 m/z displays a decrease in signal in KLA-treated samples compared to that in vehicle controls. (c) Validation of the observed decrease in butyrylLys in KLA-treated samples via western blotting of these chromatin fractions.