Method for the Determination of ¹⁵N Incorporation Percentage in Labeled Peptides and Proteins

Methods in Enzymology
Eric L Kilpatrick

Abstract

Use of labeled (15)N proteins and peptides as internal standards in isotope-dilution mass spectrometry for the quantification of proteins has been increasing and is now accepted as a gold standard for this analysis. As a necessary reagent in this process, stable heavy isotope-labeled internal standards must be rigorously characterized in a number of ways including identity, concentration, purity, and structure. Additionally, the degree of the incorporation of the heavy isotope is a critical feature to consider. For proteins that are (15)N labeled, the percentage of incorporation is a valid measurement used to assess the fitness-to-purpose of the material. This measurement should be objective, repeatable, and based on empirical analysis. One means of assigning this value is to compare a mass spectrum of the isotopic profile of a peptide against a series of theoretical profiles containing different enrichment rates. This comparison can be made using the Pearson product-moment correlation coefficient (r) to find the best match between the empirical and theoretical profiles. Theoretical profiles can be generated using probability multinomial analysis but are computationally intensive and require the use of computers for practical u...Continue Reading

Citations

Apr 4, 2017·Scientific Reports·Tao HanMingjing Tu

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