Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
Department of Biochemical Science and Technology, National Chiayi University, Chiayi 600, Taiwan.
J Am Soc Mass Spectrom. 2024 Jul 3;35(7):1507-1515. doi: 10.1021/jasms.4c00108. Epub 2024 Jun 21.
Over the past decades, proteomics has become increasingly important and a heavily discussed topic. The identification of intact proteins remains a major focus in this field. While most intact proteins are analyzed using high-resolution mass spectrometry, identifying them through low-resolution mass spectrometry continues to pose challenges. In our study, we investigated the capability of identifying various intact proteins using collision-induced dissociation (CID) and electron transfer without dissociation (ETnoD). Using myoglobin as our test protein, stable product ions were generated with CID, and the identities of the product ions were identified with ETnoD. ETnoD uses a short activation time (AcT, 5 ms) to create sequential charge-reduced precursor ion (CRI). The charges of the fragments and their sequences were determined with corresponding CRI. The product ions can be selected for subsequent CID (termed CID) combined with ETnoD for further sequence identification and validation. We refer to this method as CID/ETnoD. The use of a multistage CID activation (CID) and ETnoD protocol has been applied to several intact proteins to obtain multiple sequence identifications.
在过去的几十年中，蛋白质组学变得越来越重要，并且成为了一个备受讨论的话题。完整蛋白质的鉴定仍然是该领域的主要焦点。虽然大多数完整蛋白质都是使用高分辨率质谱分析的，但通过低分辨率质谱进行鉴定仍然具有挑战性。在我们的研究中，我们调查了使用碰撞诱导解离（CID）和无解离电子转移（ETnoD）来鉴定各种完整蛋白质的能力。使用肌红蛋白作为我们的测试蛋白，CID 产生了稳定的产物离子，并且使用 ETnoD 鉴定了产物离子的身份。ETnoD 使用短的激活时间（AcT，5 ms）来创建顺序电荷还原的前体离子（CRI）。碎片的电荷及其序列是通过相应的 CRI 确定的。产物离子可以被选择用于随后的 CID（称为 CID），并与 ETnoD 结合使用，以进行进一步的序列鉴定和验证。我们将这种方法称为 CID/ETnoD。已经将多阶段 CID 激活（CID）和 ETnoD 协议应用于几种完整蛋白质，以获得多个序列鉴定。
