Systematic evaluation of alternating CID and ETD fragmentation for phosphorylated peptides.
Systematic evaluation of alternating CID and ETD fragmentation for phosphorylated peptides.
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DOI：
10.1002/pmic.201000547
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发表时间：
2011-06
期刊：
影响因子：
3.4
通讯作者：
Pandey, Akhilesh
中科院分区：
文献类型：
--
作者：
Kim, Min-Sik;Zhong, Jun;Kandasamy, Kumaran;Delanghe, Bernard;Pandey, Akhilesh
在鸟枪式蛋白质组学中，CID已成为一种常规的多肽裂解方法，而电子转移解离(ETD)已被描述为携带不稳定的PTMS的多肽的首选方法。虽然这两种碎片技术都有其明显的优点，但它们也有各自的缺点。通过结合来自CID和ETD碎裂的数据，由于产生的碎片离子的互补性，这些缺点中的一些可能被克服。为了评估交替的CID和ETD碎裂，我们在LTQ-Orbitrap质谱仪上分析了一种复杂的磷肽混合物。当分别搜索CID和ETD派生的光谱时，我们分别从单独的CID、单独的ETD、基于决策树的CID/ETD以及交替的CID和ETD获得了2504、491、2584和3249个磷酸肽谱匹配。直观地说，在数据库搜索之前结合CID和ETD谱比单独使用任何一种方法都要好。然而，当来自交替的CID和ETD方法的光谱在数据库搜索之前被合并时，我们观察到磷酸肽-光谱匹配的数量减少。在合并CID和ETD光谱后观察到的较差的识别率反映了缺乏进行这种搜索的优化搜索算法，可能是这种方法的固有弱点。因此，尽管交替进行CID和ETD实验以鉴定磷酸肽是可取的，以增加鉴定的可信度，但在将其作为常规策略采用之前，在数据库搜索之前的合并光谱必须在各种算法的背景下进一步仔细评估。
CID has become a routine method for fragmentation of peptides in shotgun proteomics while electron transfer dissociation (ETD) has been described as a preferred method for peptides carrying labile PTMs. Though both of these fragmentation techniques have their obvious advantages, they also have their own drawbacks. By combining data from CID and ETD fragmentation, some of these disadvantages can potentially be overcome because of the complementarity of fragment ions produced. To evaluate alternating CID and ETD fragmentation, we analyzed a complex mixture of phosphopeptides on an LTQ-Orbitrap mass spectrometer. When the CID and ETD-derived spectra were searched separately, we observed 2504, 491, 2584 and 3249 phosphopeptide-spectrum matches from CID alone, ETD alone, decision tree-based CID/ETD and alternating CID and ETD, respectively. Combining CID and ETD spectra prior to database searching should, intuitively, be superior to either method alone. However, when spectra from the alternating CID and ETD method were merged prior to database searching, we observed a reduction in the number of phosphopeptide-spectrum matches. The poorer identification rates observed after merging CID and ETD spectra are a reflection of a lack of optimized search algorithms for carrying out such searches and perhaps inherent weaknesses of this approach. Thus, although alternating CID and ETD experiments for phosphopeptide identification are desirable for increasing the confidence of identifications, merging spectra prior to database search has to be carefully evaluated further in the context of the various algorithms before adopting it as a routine strategy.
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