The 15N isotope effect in Escherichia coli: A neutron can make the difference

Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the ¹⁵N isotope effect on Escherichia coli cultures that were grown in either unlabeled (¹⁴N) or ¹⁵N‐labeled media by LC‐ESI‐MS/MS‐based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between ¹⁴N and ¹⁵N‐labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between ¹⁴N and ¹⁵N‐labeled bacteria. Our data demonstrate for the first time that the introduction of the ¹⁵N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques.