Selenium-isotopic signature toward mass spectrometric identification and enzyme activity assay.

The unraveling of enzymatic reactions, especially identification of enzymatic substrates or products, is important to elucidate biological processes. Here a selenium-isotopic signature for mass spectrometric identification of enzymatic-related species is demonstrated by using selenium-containing peptides (SePeps) as substrates. Thus a strategy is proposed for rapid and precise assay of multiple enzyme activity. These SePeps can be synthesized by introduction of one selenomethionine residue in the sequence and simply identified in the full-scan mode with the feature of distinctive selenium-isotopic distribution without MS/MS verifications, which proposes a novel solution to the specific identification of enzyme-related species, allows to exclude the interferences of species with tiny mass differences in bio-samples, and meanwhile can offer a judgement on data accuracy for the analysis of enzyme activities. As a proof-of-concept, a method for multiple analysis of two representative enzymes in MCF-7 cell lysate has been developed with the isotopic peak areas of either SePep substrates or enzymatic products with the top intensities. These results could be the foundation to extend the method for more complicated enzyme systems. The selenium-isotopic signature provides a powerful protocol for high-throughput assays of peptide-metabolizing enzymes with enhanced confidence and can be extended to screen enzymatic reaction-related substrates. A selenium-isotopic signature is demonstrated for rapid mass spectrometric identification of enzyme-related species and assays of enzyme activity with enhanced confidence. Image 1 • The selenomethionine is introduced to peptide as a selenium-isotopic signature. • The peptides provide unique isotope patterns that are verified by HRMS. • The identifications of substrates and products can be performed by tracing the Se isotope in the full-scan MS mode. • This strategy provides a powerful protocol for high-throughput assays of enzymes with enhanced confidence. [ABSTRACT FROM AUTHOR]