Edinburgh Research Explorer

The Implementation and Impact of Isotope Depletion for Improved Protein Mass Spectrometry.


  • C Logan Mackay (Creator)
  • JEP Syka (Creator)
  • David Clarke (Creator)
  • DPA Kilgour (Creator)
  • Kelly Gallagher (Creator)
  • Jen Ross (Creator)

Related Edinburgh Organisations

PublisherEdinburgh DataShare
Date made available19 Aug 2020


One of the fundamental challenges of high-resolution mass spectrometry (MS) analysis of protein occurs as the molecular weight increases. Which is due to the increased chemical complexity of the protein analyte. Larger molecular weight proteins display a wider isotopologue distribution caused by an increased incorporation of heavier isotopes. This phenomenon increases the inherent complexity of a protein mass spectrum and makes achieving comprehensive analysis, like top-down fragmentation difficult. Ultimately due to the increased isotopologue overlap and reduced ion signal hindering assignment. One potential method to improve protein analysis via mass spectrometry is to apply an isotope depletion strategy. This involves recombinant expression of protein in defined growth media containing carbon and nitrogen sources depleted in heavy isotopes (notably 13C and 15N). Thus, the resulting recombinant proteins are produced with limited, heavier isotope incorporation.
An efficient and cost-effective method for isotope depletion was developed in E. coli and applied to several proteins of increasing molecular weight. After optimisation, expression in isotopically depleted media, produced recombinant protein containing 12C 99.95% and 14N 99.99%. The benefit of applying isotope depletion was assessed using top down fragmentation, mainly electron-based, techniques on both 12T FT-ICR and orbitrap Eclipse instruments.
Isotopic depletion produces simplified isotopologue distributions and increases the net contribution of the monoisotopic peak intensity to that distribution. In top-down fragmentation spectra this results in reduced overlap in fragment ion distribution and greater mass accuracy due to the presence of the abundant monoisotopic peak. Furthermore, isotopically depleted protein is observed with greater total ion signal and dramatically increased S/N. Ultimately, with these accumulative benefits, top-down fragmentation of isotopically depleted protein allows assignment of 2-3 times greater number of fragment ions. Translating into a greater sequence coverage. Particularly when the isotope depletion technique is coupled to other processes designed to increase top-down sequence coverage, like proton transfer charge reduction.
The increased number of assigned ions in isotopically depleted protein fragmentation spectra also permits a dramatic reduction in the number of averaged transients without a corresponding reduction in the sequence coverage. Permitting acquisition of greater top-down fragmentation coverage in LC-MS/MS workflows.
As the changes the isotope depletion technique causes to the isotopologue distribution are consistent, it has the potential to improve a range of protein MS analysis. This was demonstrated using isotopically depleted protein with native electrospray process, maintaining the higher order structure of protein in the gas phase. With the isotopically depleted protein it was possible to extend the feasible working mass limit of native analysis of 12T FT-ICR.

Data Citation

Gallagher, Kelly; Ross, Jennifer; Mackay, C Logan; Kilgour, DPA; Syka, JEP; Clarke, David. (2020). The Implementation and Impact of Isotope Depletion for Improved Protein Mass Spectrometry., [dataset]. University of Edinburgh. School of Chemistry. https://doi.org/10.7488/ds/2902.

ID: 163456143