Lu Jonathan, Trnka Michael J, Roh Soung-Hun, Robinson Philip J J, Shiau Carrie, Fujimori Danica Galonic, Chiu Wah, Burlingame Alma L, Guan Shenheng
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA.
Princeton University, Princeton, NJ, 08544, USA.
J Am Soc Mass Spectrom. 2015 Dec;26(12):2141-51. doi: 10.1007/s13361-015-1235-6. Epub 2015 Sep 1.
Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.
天然电喷雾电离质谱（天然质谱）在能保留蛋白质三级和四级结构大部分特征的条件下测量生物分子，从而能够直接表征完整的大型蛋白质组装体。然而，由于电喷雾过程后残留的溶剂化和加合作用，源自这些组装体的天然质谱图常常部分被低信噪比以及宽峰形所掩盖。宽峰宽以及来自高电荷离子的连续电荷态系列紧密间隔这一事实意味着，包含多种物种的天然质谱图常常存在高度的峰重叠，或者包含高度交错的电荷包络。这种情况给峰检测、正确的电荷态和电荷包络归属以及最终提取所研究的非共价组装体的相关基础质量值带来了挑战。在本报告中，我们描述了一种为解决天然质谱中的峰检测、峰重叠和电荷态归属问题而开发的综合算法，称为PeakSeeker。通过检查原始质谱的二阶导数来检测重叠峰。通过将电荷包络的线性组合拟合到整体实验质谱来确定分子物种的电荷态分布。如对（1）含有严重重叠峰的合成单核小体、（2）具有许多紧密交错离子信号的RNA聚合酶II/α-鹅膏蕈碱复合物以及（3）含有高水平背景噪声的人TriC复合物的分析所示，该软件能够对大型蛋白质组装体的异质、复杂且有噪声的天然质谱进行去卷积。图形摘要ᅟ。
