Improved Discrimination of Asymmetric and Symmetric Arginine Dimethylation by Optimization of the Normalized Collision Energy in Liquid Chromatography–Mass Spectrometry Proteomics

Abstract

Protein arginine methylation regulates diverse biological processes including signaling, metabolism, splicing, and transcription. Despite its important biological roles, arginine methylation remains an understudied post-translational modification. Partly, this is because the two forms of arginine dimethylation, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), are isobaric and therefore indistinguishable by traditional mass spectrometry techniques. Thus, there exists a need for methods that can differentiate these two modifications. Recently, it has been shown that the ADMA and SDMA can be distinguished by the characteristic neutral loss (NL) of dimethylamine and methylamine, respectively. However, the utility of this method is limited because the vast majority of dimethylarginine peptides do not generate measurable NL ions. Here, we report that increasing the normalized collision energy (NCE) in a higher-energy collisional dissociation (HCD) cell increases the generation of the characteristic NL that distinguish ADMA and SDMA. By analyzing both synthetic and endogenous methyl-peptides, we identify an optimal NCE value that maximizes NL generation and simultaneously improves methyl-peptide identification. Using two orthogonal methyl peptide enrichment strategies, high pH strong cation exchange (SCX) and immunoaffinity purification (IAP), we demonstrate that the optimal NCE increases improves NL-based ADMA and SDMA annotation and methyl peptide identifications by 125% and 17%, respectively, compared to the standard NCE. This simple parameter change will greatly facilitate the identification and annotation of ADMA and SDMA in mass spectrometry-based methyl-proteomics to improve our understanding of how these modifications differentially regulate protein function.

Multi-OMICs analysis reveals metabolic and epigenetic changes associated with macrophage polarization

“…4 C ). Based on mass spectrometry (MS), arginine dimethylation was predominately asymmetric because the MH + -45 ion (loss of dimethylamine) was detected but the symmetric demethylation signature MH + -31 ion (loss of methylamine) was not ( 53 ). Interestingly monomethylated arginine, also known as N-mono-methyl-L-arginine, and asymmetric dimethylated arginine are known to modulate NO production by competing with arginine for NOS binding ( 54 ).…”

Mesh Fragmentation Improves Dissociation Efficiency in Top-down Proteomics

“…We evaluated the yield of matched fragment ions for the same proteoform across different charge states and different normalized collision energies (NCE) using higher-energy collisional dissociation (HCD). NCE is defined as a single energy value provided in Orbitrap mass spectrometer HCD MS/MS experiments . DDA is often coupled with HCD, one of the most commonly used fragmentation methods in top-down proteomics. ,, Three standard proteins, including ubiquitin, cytochrome C, and myoglobin, each exhibiting unique charge state distributions, were examined for this study (Figure ).…”

“…NCE is defined as a single energy value provided in Orbitrap mass spectrometer HCD MS/MS experiments. 33 DDA is often coupled with HCD, one of the most commonly used fragmentation methods in topdown proteomics. 2,6,8 Three standard proteins, including ubiquitin, cytochrome C, and myoglobin, each exhibiting unique charge state distributions, were examined for this study (Figure 1).…”

A Chemical Acetylation-Based Mass Spectrometry Platform for Histone Methylation Profiling

“…While the need for a higher collision energy might be specific for this peptide, optimization of the collision energy is necessary for best neutral loss production. The need for higher collision energy for better production for the ADMA/SDMA diagnostic ions has recently been reported as a more general rule ( 40 ). Monitoring the neutral loss fragments from ions b4–b6, we could clearly discriminate between H4R3 ADMA and SDMA ( supplemental Fig.…”