Determination of Isotope-Enrichment Ratios in Proteins by High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Abstract

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry has been applied for determining the ¹³C and ¹⁵N stable isotope content of a 10 kDa protein molecule. Purified samples of modified human ubiquitin, expressed in E. coli grown in a ¹³C and ¹³N isotope-enriched medium, are electrosprayed in a 4.7 T FT-ICR instrument. The intensity profiles of the isotopically-resolved envelope of the molecular ions in different charge states are averaged. The experimental data are then compared to theoretically-generated isotopic profiles for the same molecule at varying ratios of ¹²C: ¹³C and ¹⁴N: ¹⁵N isotope enrichment. The isotope-enrichment ratios giving the best overlap between simulated and experimental data correspond to estimates of incorporation from the ¹³C and ¹⁵N isotopes present in the microorganism growth medium.