Tracking internal and external ions for constrained peptides leads to enhanced sequence coverage and disulfide bond deciphering
Tracking internal and external ions for constrained peptides leads to enhanced sequence coverage and disulfide bond deciphering
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DOI：
10.1016/j.jpba.2021.113893
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发表时间：
2021-01-11
影响因子：
3.4
通讯作者：
Crittenden, Christopher M.
中科院分区：
文献类型：
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作者：
Chin, Steven;Chen, Tao;Crittenden, Christopher M.
由于限制和保护的氨基酸骨架，富含二硫键的肽和蛋白质的自上而下的表征提出了许多挑战。通常，由于与这些生物分子的完整、自上而下分析相关的挑战，需要化学还原来还原二硫键和/或利用酶消化（自下而上分析）在质谱分析之前选择性地切割氨基酸序列。虽然过度的自上而下的表征技术，如紫外光解离（UVPD）或电子捕获解离（ECD），已经证明了在自上而下的工作流程中断裂二硫键的能力，但这些技术的实施和所得碎片光谱的分析并不简单，许多实验室和用户通常无法获得。在本文提出的研究中，进行富含二硫键的肽的传统碰撞诱导解离（CID）以确认二硫键连接性并定位这些合成治疗肽的化学修饰。虽然碰撞活化不会在二硫键键区域内线性裂解肽骨架（典型的N-和C-末端碎片离子），但通过二级裂解途径在整个序列中始终产生内部和外部离子。在本研究中，对具有相似二硫键连接性但氨基酸组成不同的七种富含二硫键的肽（肽A-G）进行碰撞活化，以进行测序和二硫键确认。虽然这些肽仅产生四个线性B-和y-型碎片离子，但在搜索内部和外部碎片离子时观察到整个氨基酸序列的碎片化。这些离子通常在传统的自上而下测序实验中不被考虑，这是由于具有用于碎片离子识别的增加的搜索空间的计算挑战。通过鉴定可重现的内部和外部碎片离子，还可以定位位点特异性修饰，例如肽A中第18个残基上的氧化。最终，这种内部和外部离子的观察和识别简化了准确描述这些传统上具有挑战性的生物分子的二硫键连接和测序所需的实验过程和湿化学。在自上而下的完整肽和蛋白质分析中，应进一步考虑这些非传统碎片离子，特别是当涉及非线性序列时。(C)2021爱思唯尔有限公司版权所有。
Top-down characterization of disulfide-rich peptides and proteins presents many challenges due to the constrained and protected amino acid backbone. Typically, chemical reduction is required to reduce the disulfide bonds and/or enzymatic digestion (bottom-up analysis) is utilized to selectively cleave the amino acid sequence prior to mass spectrometry analysis owing to the challenges associated with intact, top-down analysis of these biomolecules. While extravagant top-down characterization techniques such as ultraviolet photodissociation (UVPD) or electron capture dissociation (ECD), have demonstrated the ability to break disulfide bonds in top-down workflows, implementation of these technologies and analysis of the resulting fragmentation spectra is not trivial and often inaccessible to many laboratories and users. In the study presented herein, traditional collision induced dissociation (CID) of disulfide-rich peptides is performed to confirm the disulfide bond connectivity and localize chemical modifications for these synthetic therapeutic peptides. While collisional activation does not fragment the peptide backbone linearly (typical N- and C-terminal fragment ions) within the disulfide-bonded regions, internal and external ions are consistently produced throughout the sequence via secondary fragmentation pathways. In this study, seven disulfide-rich peptides (Peptides A - G) with similar disulfide connectivity but varying amino acid composition were subjected to collisional activation for sequencing and disulfide bond confirmation. While only four linear b- and y-type fragment ions are produced for these peptides, fragmentation throughout the amino acid sequence is observed when searching for internal and external fragment ions. These ions are typically not considered during traditional top-down sequencing experiments due to the computational challenge of having an increased search space for fragment ion identification. Through the identification of reproducible internal and external fragment ions, site-specific modifications can also be localized, such as oxidation on the 18th residue in Peptide A. Ultimately, this observation and identification of internal and external ions simplifies the experimental process and wet-chemistry required to accurately depict the disulfide connectivity and the sequencing of these traditionally challenging biomolecules. Further consideration to these non-traditional fragment ions should be given during top-down intact peptide and protein analysis, especially when non-linear sequences are involved. (C) 2021 Elsevier B.V. All rights reserved.