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

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.

蛋白质的高分辨质谱（MS）分析面临的核心挑战之一，随蛋白质分子量升高而愈发凸显，其根源在于蛋白质分析物的化学复杂性随分子量增加而提升。分子量更大的蛋白质，因更易掺入重同位素，其同位素异构体分布范围更广。该现象会加剧蛋白质质谱的本征复杂度，使得自上而下碎裂等全面分析难以实现，究其根本，是同位素异构体的重叠程度升高、离子信号被削弱，阻碍了谱峰归属。

一种有望通过质谱技术改善蛋白质分析的策略，是采用同位素耗竭法。该方法指在含有耗竭了重同位素（尤其是¹³C和¹⁵N）的碳源与氮源的限定成分培养基中，对蛋白质进行重组表达，由此得到的重组蛋白质仅会掺入极少量重同位素。

研究人员在大肠杆菌（E. coli）中开发了一种高效且经济的同位素耗竭方法，并将其应用于一系列分子量递增的蛋白质。经优化后，在同位素耗竭培养基中表达得到的重组蛋白质，其¹²C占比达99.95%，¹⁴N占比达99.99%。

研究人员借助12T傅里叶变换离子回旋共振（FT-ICR）与轨道阱Eclipse质谱仪，主要通过电子诱导碎裂类技术开展自上而下碎裂分析，以此评估同位素耗竭法的应用优势。同位素耗竭可简化同位素异构体分布，提升单同位素峰强度在整体分布中的净占比。在自上而下碎裂谱图中，这一效果可减少碎片离子分布的重叠程度，并因丰度更高的单同位素峰的存在，提升质谱质量准确度。

此外，同位素耗竭的蛋白质可获得更高的总离子信号，且信噪比（S/N）显著提升。综合上述优势，对同位素耗竭蛋白质进行自上而下碎裂，可归属的碎片离子数量提升2~3倍，进而实现更高的序列覆盖度。尤其当同位素耗竭技术与其他可提升自上而下序列覆盖度的手段（如质子转移电荷还原）联用时，该效果更为显著。

同位素耗竭蛋白质的碎裂谱图中可归属离子数量的提升，还允许在不降低序列覆盖度的前提下，大幅减少需平均的瞬态信号次数，从而在液相色谱-串联质谱（LC-MS/MS）流程中实现更高的自上而下碎裂覆盖度。

由于同位素耗竭技术对同位素异构体分布的改变效果稳定，其有望改善多种蛋白质质谱分析场景。研究人员通过将同位素耗竭蛋白质与天然态电喷雾流程联用，在气相中保留蛋白质的高级结构，验证了这一潜力：借助同位素耗竭蛋白质，可将12T傅里叶变换离子回旋共振（FT-ICR）质谱的天然态分析可行质量上限进一步拓展。