Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS.
Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS.
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DOI：
10.1021/ac025807j
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发表时间：
2002-08
影响因子：
7.4
通讯作者：
V. Schnaible;S. Wefing;A. Resemann;D. Suckau;A. Bücker;Sybille Wolf-Kümmeth;D. Hoffmann
中科院分区：
文献类型：
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作者：
V. Schnaible;S. Wefing;A. Resemann;D. Suckau;A. Bücker;Sybille Wolf-Kümmeth;D. Hoffmann
提出了一种自动筛选方法，该方法使用二硫键的 MALDI 源内衰变 (MALDI-ISD) 来鉴定 MALDI 质谱中的二硫键连接的肽。由二硫键的 ISD 释放的肽可以以与半胱氨酸残基减少的单质子化肽相对应的 m/z 比进行检测。因此，筛选满足方程 m/z（峰 A）+ m/z（峰 B）- m/z（H2 + H+）= m/z（峰 C）的信号模式的峰列表，有助于识别二硫键连接肽（峰 A 和 B）及其前体（峰 C）的推定 ISD 片段。来自推定二硫键连接肽的信号（峰 C）经过 LIFT-TOF/TOF-MS 验证，以确认二硫键的存在。使用这种方法，我们鉴定了 RNAseA 中的所有 4 个二硫键和 8 个双二硫键簇，其中包含 BSA 中 17 个二硫键中的 16 个。所提出的筛选方法加速了 RNAseA 和 BSA 中二硫键的鉴定，因为要获取的 MS/MS 谱图数量减少了 1 个数量级。为 LIFT-TOF/TOF-MS 选择的信号中不到 5% 不对应于二硫键连接的肽。此外，二硫键连接肽的可能分配数量减少了 2-3 个数量级，因为 ISD 对二硫键断裂机制的了解允许使用更严格的规则来解释质谱。因此，与传统方法相比，二硫键连接肽的 MS/MS 谱图的解释大大简化。
An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) - m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2-3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/ MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.