New chemical strategy boosts accuracy in proteomics

Overlabeling of peptides in proteomics mass spectrometry reduces protein identification and quantitation precision. In proteomics sample preparation, a chemical called N-hydroxysuccinimide, or NHS, is commonly used in tagging proteins to identify proteins for quantitation. However, NHS can also react with other amino acid residues, forming unwanted O-ester derivatives, which bond to oxygen atoms of amino acids such as serine, tyrosine and threonine, complicating analysis. While existing methods, such as hydroxylamine treatment, have been used to reduce excess NHS esters, it is not fully effective, and scientists lack methods to sufficiently remove overlabeled peptides.

Yana Demyanenko and a team of researchers based in the U.K. and Germany published an article in Molecular & Cellular Proteomics, where they developed a methylamine-based method to remove these O-ester modifications. They found that methylamine was the most effective in reducing overlabeled peptides among various tested reagents, such as hydroxylamine, O-methoxylamine HCl, hydrazine hydrate, Tris and ammonium hydroxide. In contrast, standard labeling without quenching led to over 25% overlabeled peptides, while hydroxylamine treatment reduced this to only 10%. Methylamine, however, reduced the overlabeling to less than 1% without affecting the labeling rate or causing additional modifications. Future research will apply this methylamine-based approach to different proteomics workflows to improve peptide identification and quantitation.