Comparison of data-acquisition methods for the identification and quantification of histone post-translational modifications on a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer
Comparison of data-acquisition methods for the identification and quantification of histone post-translational modifications on a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer
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DOI：
10.1002/rcm.8401
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发表时间：
2019-05-30
影响因子：
2
通讯作者：
Dickman, Mark J.
中科院分区：
文献类型：
--
作者：
Cole, Joby;Hanson, Eleanor J.;Dickman, Mark J.
组蛋白翻译后修饰在真核生物基因表达调控中起着重要作用。质谱（MS）已经成为表征和定量组蛋白PTM的有力方法，因为它允许无偏地鉴定和定量多个组蛋白PTM，包括存在的修饰的组合。方法在这项研究中，我们比较了一系列的数据采集方法的识别和定量的组蛋白PTM使用Q Exactive HF Orbitrap。在MS 2分辨率为120 K、60 K、30 K的条件下，比较了三种不同的数据相关分析（DDA）方法。我们还比较了一系列使用20 m/z的MS 2分离窗口和DIAvw的数据独立分析（DIA）方法，以鉴定和定量中国仓鼠卵巢（CHO）细胞中的组蛋白PTM。结果较低分辨率方法提供的MS 2扫描数量增加导致查询、肽序列匹配（PSM）数量增加，Mascot Ion评分大于46的肽蛋白质型数量增加。没有观察到Delta评分>17的肽蛋白形式的比例的差异。与其他数据采集方法相比，在DIA MS 1 60 K MS 2 30 K 20 m/z隔离窗口中获得了较低的变异系数（CV）。结论DIA在组蛋白PTM的分析中具有灵活性强、能识别同量异序肽的优点，与DDA具有相同的性能。我们能够识别71个组蛋白H3和H4的修饰组蛋白肽，并在每种不同的采集方法中定量64个。
Rationale Histone post-translational modifications (PTMs) play key roles in regulating eukaryotic gene expression. Mass spectrometry (MS) has emerged as a powerful method to characterize and quantify histone PTMs as it allows unbiased identification and quantification of multiple histone PTMs including combinations of the modifications present. Methods In this study we compared a range of data-acquisition methods for the identification and quantification of the histone PTMs using a Q Exactive HF Orbitrap. We compared three different data-dependent analysis (DDA) methods with MS2 resolutions of 120K, 60K, 30K. We also compared a range of data-independent analysis (DIA) methods using MS2 isolation windows of 20 m/z and DIAvw to identify and quantify histone PTMs in Chinese hamster ovary (CHO) cells. Results The increased number of MS2 scans afforded by the lower resolution methods resulted in a higher number of queries, peptide sequence matches (PSMs) and a higher number of peptide proteoforms identified with a Mascot Ion score greater than 46. No difference in the proportion of peptide proteoforms with Delta scores >17 was observed. Lower coefficients of variation (CVs) were obtained in the DIA MS1 60 K MS2 30 K 20 m/z isolation windows compared with the other data-acquisition methods. Conclusions We observed that DIA which offers advantages in flexibility and identification of isobaric peptide proteoforms performs as well as DDA in the analysis of histone PTMs. We were able to identify 71 modified histone peptides for histone H3 and H4 and quantified 64 across each of the different acquisition methods.