Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433-434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in 13C and 15N and demonstrating its advantages for top-down analysis of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experiments-highlighting advantages for top-down LC-MS/MS workflows and top-down proteomics.

中文翻译：

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同位素消耗质谱（ID-MS），用于精确测定质量和改善完整蛋白的自上而下的序列覆盖率。

自上而下的质谱（MS）是一种越来越重要的蛋白质表征技术。但是，在许多生物学MS实验中，自上而下方法的实用性在较高分子量下仍然受到限制。在很大程度上，这是由于随着分子质量的增加将质谱信号划分为越来越多的同位素峰而产生的有害影响。20年前首次报道了通过消耗重的稳定同位素来减少蛋白质的同位素同位素分布（Marshall，AG; Senko，MW; Li，W .; Li，M .; Dillon，S.，Guan，S .; Logan，通过13 C，15N双重耗尽和FT-ICR质谱分析（TM。蛋白分子质量达到1 Da）（J. Am。Chem。Soc。1997，119，433-434。），并且已经证明了几种小蛋白。在这里，我们扩展这种方法，引入了一种新的高效方法来生产在13C和15N中消耗的重组蛋白，并证明了其对自上而下分析较大蛋白（约50 kDa）的优势。同位素耗尽的蛋白质的FT-ICR MS显示同位素分布大大降低，观察到高达50 kDa的单同位素信号。在自上而下的碎片实验中，降低的光谱复杂度减轻了碎片离子信号的重叠，单同位素信号的存在允许以更高的质量精度进行分配，信噪比的显着提高（最高7倍）可以极大地提高减少获取时间。与具有天然同位素丰度的蛋白质的可比分析相比，这些复合优势使碎片离子的分配量增加了约3倍。最后，