Data-dependent electron capture dissociation FT-ICR mass spectrometry for proteomic analyses
Data-dependent electron capture dissociation FT-ICR mass spectrometry for proteomic analyses
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DOI：
10.1021/pr050090c
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发表时间：
2005-09-01
影响因子：
4.4
通讯作者：
Zeller, M
中科院分区：
文献类型：
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作者：
Cooper, HJ;Akbarzadeh, S;Zeller, M
电子捕获解离(ECD)技术在多肽和蛋白质的分析中具有许多优点，因此在蛋白质组学领域显示出巨大的潜力。最近的发展已将ECD所需的时间减少到毫秒，从而使该技术与在线分离技术，如高效液相色谱兼容。在这里，我们展示了结合电子捕获到高通量的数据依赖的LC-MS/MS分析蛋白质样本的方法。该方法被应用于从软骨细胞中分离的蛋白质Fc-ROR2的分析，是此类样品的LC-ECD-MS/MS的第一个例子。蛋白质序列覆盖率为29%。在这一覆盖范围内，分离了15个多肽并进行了ECD。在大多数情况下，ECD产生的序列标签超过70%(根据肽骨架裂解的数量)。使用SEQUEST算法在非冗余的人类NCBI数据库中搜索ECD数据。指定了ROR2蛋白和Ig G(Fc结构域)。这些结果证明了ECD作为高通量蛋白质组策略中的一种完整技术的适用性。
Electron capture dissociation (ECD) offers many benefits for the analysis of peptides and proteins, and consequently shows great potential for the field of proteomics. Recent developments have reduced the time scale required for ECD to milliseconds resulting in the technique's compatibility with on-line separation techniques, e.g., HPLC. Here, we demonstrate incorporation of ECD into a high-throughput data-dependent LC-MS/MS approach for the analysis of proteomic samples. The approach is applied to analysis of the protein Fc-ROR2 isolated from chondrocytes and is the first example of LC-ECD-MS/MS of such a sample. Protein sequence coverage was 29%. Within that coverage, fifteen peptides were isolated and subjected to ECD. In most cases, the sequence tag generated by ECD was over 70% (in terms of the number of peptide backbone cleavages). The ECD data were searched against the nonredundant human NCBI database using the SEQUEST algorithm. Protein ROR2 was assigned, as was IgG (Fc domain). The results demonstrate the suitability of ECD as an integral technique in high-throughput proteomic strategies.