Implementation and impact of isotope depletion for improved protein mass spectrometry
Gallagher, Kelly Jane
One of the fundamental challenges of high-resolution mass spectrometry (MS) analysis of protein occurs as the molecular weight increases. This is due to the increased chemical complexity of the protein analyte, as larger molecular weight proteins display a wider isotopologue distribution caused by increased incorporation of heavier isotopes. This phenomenon increases the inherent complexity of the protein mass spectrum. In top-down fragmentation workflows, this is observed as densely packed spectral regions of fragment ions with heavily overlapping isotopologue distributions. It also associated with a reduction in fragment ion signal, all of which contributes to hinder ion assignment. One potential method to improve protein analysis via mass spectrometry is to apply an isotope depletion strategy. This involves the recombinant expression of protein within a defined growth media, containing carbon and nitrogen sources depleted in heavy isotopes (notably 13C and 15N). Thus, the resulting recombinant proteins are expressed 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, the recombinant protein was expressed typically containing isotope abundances of 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. The change to the protein mass spectrum, when using the isotopic depletion strategy, is observed as a simplified isotopologue distribution; an increased net contribution of the monoisotopic peak intensity and an increase of the fragment ion signal. 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. As a result of these accumulative benefits, top-down fragmentation of isotopically depleted protein allows assignment of 2-3 times greater number of fragment ions. This-in-turn allows attainment of higher sequence coverage, particularly when the isotope depletion strategy is used in conjunction with 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. Therefore the isotope depletion strategy facilitates the acquisition of higher top-down fragmentation sequence coverage in LC-MS/MS workflows. As the isotope depletion strategy causes consistent simplification to the isotopologue distribution, it has the potential to improve a range of protein MS analysis. This was further demonstrated using isotopically depleted protein with native protein analysis, which maintains the higher-order structure of proteins within 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.