State Key Laboratory of Molecular Reaction Dynamics, Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Liaoning, China.
Rapid Commun Mass Spectrom. 2013 Jun 15;27(11):1267-77. doi: 10.1002/rcm.6565.
Soft ionization, high-resolution mass spectrometry is widely used to characterize large biological molecules, such as proteins. Deconvolution ('deisotoping') of isotopic envelopes (iEs) in biological mass spectra into monoisotopic or average masses is challenging due to low signals and heavily overlapped iEs, resulting in many wrong interpretations.
Isotopic envelopes (iEs) are directly used without deisotoping to identify biological molecules. An algorithm, isotopic mass-to-charge ratio (m/z) and envelope fingerprinting (iMEF), was implemented in the ProteinGoggle search engine for top-down intact protein database searching. iMEF combines isotopic m/z fingerprinting (iMF) and isotopic envelop fingerprinting (iEF), where 'Isotopic mass-to-charge ratio' means the m/z value of the most abundant isotopic peak within the iE of a precursor or product ion. iMF is used to 'fish' precursor or product ion candidates from the database, which is pre-built and contains all iE information (precursor and product ions) of all proteoforms of the studied system. iEF identifies matching precursor or product ions. A protein is finally identified with user-specified total number of matching product ions and post-translational modification scores.
The working principles of iMEF and ProteinGoggle, and the definition of a set of related parameters and scoring metrics, are illustrated with high-resolution tandem mass spectrometric analysis of a mixture of ubiquitin and the HUMAN histone H4 proteoforms. Ubiquitin was confidently identified from its CID, ETD, and HCD spectra with 57, 91, and 66 matching product ions, respectively; 125 proteoforms were confidently found from the H4 dataset. The locations of PTMs in 54 and 6 isoforms were partially and fully identified.
Database search with iMEF bypasses 'deisotoping' avoiding associated errors, and also provides full quality control of matching precursor and product ions and finally protein IDs. Overlapped iEs of different product ions could also be confidently unwrapped in situ. Improvement and addition of more functionalities and utilities of ProteinGoggle are underway.
软电离、高分辨率质谱广泛用于对大生物分子（如蛋白质）进行特征分析。由于信号低且同位素包络（iE）严重重叠，生物质谱中的同位素包络（iE）的解卷积（“去同位素峰”）将其转化为单同位素或平均质量极具挑战性，这会导致许多错误的解释。
不进行去同位素峰处理，直接使用同位素包络（iE）来识别生物分子。一种算法，即同位素质荷比（m/z）和包络指纹识别（iMEF），已在 ProteinGoggle 搜索引擎中实现，用于自上而下的完整蛋白质数据库搜索。iMEF 结合了同位素 m/z 指纹识别（iMF）和同位素包络指纹识别（iEF），其中“同位素质荷比”是指 iE 中前体或产物离子最丰富同位素峰的 m/z 值。iMF 用于从数据库中“钓取”前体或产物离子候选物，该数据库是预先构建的，包含研究系统中所有蛋白形式的所有 iE 信息（前体和产物离子）。iEF 用于识别匹配的前体或产物离子。最后，根据用户指定的匹配产物离子总数和翻译后修饰分数来鉴定蛋白质。
通过对泛素和 HUMAN 组蛋白 H4 蛋白形式混合物的高分辨率串联质谱分析，说明了 iMEF 和 ProteinGoggle 的工作原理以及一组相关参数和评分指标的定义。使用 CID、ETD 和 HCD 谱，分别有 57、91 和 66 个匹配产物离子可靠地鉴定出泛素；从 H4 数据集可靠地发现了 125 种蛋白形式。54 种和 6 种异构体中部分和完全鉴定出 PTM 的位置。
使用 iMEF 进行数据库搜索可绕过“去同位素峰”处理，避免相关错误，还可提供匹配前体和产物离子以及最终蛋白质 ID 的全面质量控制。不同产物离子的重叠 iE 也可以在原位有信心地展开。目前正在改进和添加更多的功能和 ProteinGoggle 的实用程序。
