Highly Efficient Separation of Methylated Peptides Utilizing Selective Complexation between Lysine and 18-Crown-6

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Protein methylation is a crucial post-translational modification involved in various cellular processes.
• Separating methylated peptides is challenging due to low abundance and subtle differences from nonmethylated counterparts.

Purpose of the Study:

• To develop a simple and highly efficient method for separating lysine methylated and nonmethylated peptides.
• To improve the enrichment and identification of methylated peptides in complex biological samples.

Main Methods:

• Utilized 18-crown-6 as a mobile phase additive in high-performance liquid chromatography (HPLC).
• Employed selective complexation between lysine and 18-crown-6 to enhance peptide retention on a C18 stationary phase.
• Verified binding mechanisms using NMR titration, biolayer interferometry, and quantum chemistry calculations.

Main Results:

• Achieved excellent baseline separation between lysine methylated and nonmethylated peptides.
• Successfully enriched and separated four methylated peptides from a complex sample with significantly improved signal-to-noise ratios.
• Demonstrated effective identification of histone methylation information using the developed enrichment method.

Conclusions:

• The 18-crown-6 based HPLC method provides a novel and effective approach for lysine methylated peptide enrichment.
• This technique offers a valuable tool for advancing post-translational modification proteomics.
• The study opens new avenues for selective enrichment strategies in proteomics.