Biomolecular Mass Spectrometry and Proteomics Group, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
J Proteome Res. 2011 Sep 2;10(9):4334-41. doi: 10.1021/pr200156b. Epub 2011 Jul 21.
In proteomics, selected reaction monitoring (SRM) is rapidly gaining importance for targeted protein quantification. The triple quadrupole mass analyzers used in SRM assays allow for levels of specificity and sensitivity hard to accomplish by more standard shotgun proteomics experiments. Often, an SRM assay is built by in silico prediction of transitions and/or extraction of peptide precursor and fragment ions from a spectral library. Spectral libraries are typically generated from nonideal ion trap based shotgun proteomics experiments or synthetic peptide libraries, consuming considerable time and effort. Here, we investigate the usability of beam type CID (or "higher energy CID" (HCD)) peptide fragmentation spectra, as acquired using an Orbitrap Velos, to facilitate SRM assay development. Therefore, peptide fragmentation spectra, obtained by ion-trap CID, triple-quadrupole CID (QqQ-CID) and Orbitrap HCD, originating from digested cellular lysates, were compared. Spectral comparison and a dedicated correlation algorithm indicated significantly higher similarity between QqQ-CID and HCD fragmentation spectra than between QqQ-CID and ion trap-CID spectra. SRM transitions generated using a constructed HCD spectral library increased SRM assay sensitivity up to 2-fold, when compared to the use of a library created from more conventionally used ion trap-CID spectra, showing that HCD spectra can assist SRM assay development.
在蛋白质组学中，选择反应监测（SRM）技术因其能够靶向定量蛋白质而迅速受到重视。三重四极杆质谱仪在 SRM 分析中被广泛应用，其特异性和灵敏度水平是更标准的鸟枪法蛋白质组学实验难以实现的。通常，通过计算机预测转换和/或从光谱库中提取肽前体和片段离子来构建 SRM 分析。光谱库通常是从不理想的基于离子阱的鸟枪法蛋白质组学实验或合成肽库中生成的，需要耗费大量的时间和精力。在这里，我们研究了使用轨道阱 Velos 获得的束流型 CID（或“更高能量 CID”（HCD））肽碎裂谱在促进 SRM 分析开发方面的可用性。因此，我们比较了来源于细胞裂解物的离子阱 CID、三重四极杆 CID（QqQ-CID）和轨道阱 HCD 获得的肽碎裂谱。光谱比较和专用相关算法表明，QqQ-CID 和 HCD 碎裂光谱之间的相似度明显高于 QqQ-CID 和离子阱-CID 光谱之间的相似度。与使用更传统的离子阱-CID 光谱创建的库相比，使用构建的 HCD 光谱库生成的 SRM 转换可将 SRM 分析的灵敏度提高 2 倍，这表明 HCD 光谱可以辅助 SRM 分析开发。
