Systematic approach to group-specific isotopic labeling of proteins for vibrational spectroscopy

Group-specific isotopic labeling of proteins using stable isotopes such as 15N, 13C, or 2H is a widely applied method for the accurate assignment of signals in FTIR spectroscopy. In this work, we report a systematic study on the metabolic incorporation of various stable isotope-coded amino acids into proteins of interest during organism growth. Target proteins were overexpressed in Escherichia coli cells that were grown in modified M9 medium supplemented with the respective stable isotope-coded amino acid. Through state of the art mass spectrometric analyses by means of in-gel tryptic digestion, nano-reversed-phase liquid chromatography and electrospray ionization tandem mass spectrometry we were able to quantify the degree of both the incorporation and the spreading of the isotopic label into target proteins in a fast and efficient manner. Our analyses show that specificity and/or efficiency of such metabolic labeling experiments are often limited. The efficient incorporation of stable isotopes can be facilitated by use of E. coli strains auxotrophic for the respective isotope-coded amino acid. We tested various auxotrophic strains for their potential to yield specifically labeled proteins suitable for FTIR or Raman measurements and optimized the culturing conditions. As an application example of our method we show the assignment of tyrosine bands in the time-resolved difference spectrum of the GTPase reaction of Ras by use of group-specific isotopic labeling. In conclusion, our work provides a valuable guideline for the production of group-specific isotopically labeled proteins for FTIR spectroscopic analysis.