Beyond quantitative proteomics:: Signal enhancement of the a1 ion as a mass tag for peptide sequencing using dimethyl labeling
Beyond quantitative proteomics:: Signal enhancement of the a1 ion as a mass tag for peptide sequencing using dimethyl labeling
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DOI：
10.1021/pr049837
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发表时间：
2005-01-01
影响因子：
4.4
通讯作者：
Chen, SH
中科院分区：
文献类型：
--
作者：
Hsu, JL;Huang, SY;Chen, SH
稳定的基于同位素的二甲基标记，其通过还原甲基化产生二甲基标记的末端胺或单甲基化的脯氨酸N-末端（Anal. 2003，75，6843-6852）被报道为用于全球定量蛋白质组学的有前景的策略，因为该方法简单且反应快速且完全。这种标记策略为产生的α，离子提供了信号增强，这通常很难在大多数非衍生化片段中检测到。为了辅助肽测序，在本研究中，在碰撞诱导解离（CID）或源后衰变（PSD）模式下产生的增强的α 1离子被进一步表征并作为质量标签用于对N-末端氨基酸的身份进行指纹识别。通过对标准肽、血红蛋白胰蛋白酶解物和细胞裂解物的分析，证明了这种信号增强作用发生在所有20个氨基酸残基的α_1离子上，并根据稳定的季铵离子的形成解释了这种现象。α 1离子的准确测定显示出增加肽从头测序的机会，并且还在通过数据库搜索鉴定蛋白质时获得的分数中提供更高的置信度。此外，α 1离子被进一步证明可用作Q-TOF仪器中前体离子扫描的通用标签，导致测序的肽离子数量更大。结合差异定量的能力，稳定的同位素为基础的二甲基标记增加了标记方法的MS为基础的蛋白质组学的有用性。
Stable isotope-based dimethyl labeling that produces a dimethyl labeled terminal amine or a monomethylated proline N-terminus by reductive methylation (Anal. Chem. 2003, 75, 6843-6852) was reported as a promising strategy for global quantitative proteomics because of the simplicity of the process and its fast and complete reaction. This labeling strategy provides a signal enhancement for the produced a, ions, which are usually hard to detect among most of the nonderivatized fragments. To assist peptide sequencing, in this study, the enhanced a, ion produced under either collision induced dissociation (CID) or post source decay (PSD) modes was further characterized and applied as a mass tag for fingerprinting the identity of N-terminal amino acid. On the basis of the analysis of standard peptides, tryptic digests of hemoglobin and cell lysates, it was proved that such signal enhancement occurred to a, ions derived from all 20 of the amino acids residues and this phenomenon was explained based the formation of stable quaternary immoniun ions. Accurate determination of a, ions was shown to increase the chance for peptide de novo sequencing and also provided higher confidence in the scores obtained when identifying a protein through database searching. In addition, the a, ion was further demonstrated to be used as a universal tag for precursor ion scan in a Q-TOF instrument, leading to a greater number of peptide ions sequenced. Combined with the capability for differential quantitation, the stable isotope-based dimethyl labeling increases the usefulness of the labeling method for MS-based proteomics.