Method for the determination of 15N incorporation percentage in labeled peptides and proteins
Eric L. Kilpatrick
Use of labeled 15N 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 quality of the incorporation of the heavy isotope is a critical feature to consider. For proteins that are 15N 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 a different enrichment rates. This comparison can be made using the Pearson 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 use. The method described in this chapter describes the development and use a computer program to calculate the percentage of 15N enrichment of a labeled internal standard. Additionally, methods will be described for the empirical determination of an isotopic profile using a variety of mass spectrometry techniques.
Isotope labeling of biomolecules, Part A and Part B