Department of Chemistry, University of California, Riverside, California 92521, United States.
J Am Soc Mass Spectrom. 2024 Apr 3;35(4):705-713. doi: 10.1021/jasms.3c00373. Epub 2024 Mar 5.
Isomerized amino acid residues have been identified in many peptides extracted from tissues or excretions of humans and animals. These isomerized residues can play key roles by affecting biological activity or by exerting an influence on the process of aging. Isomerization occurs spontaneously and does not result in a mass shift. Thus, identifying and localizing isomerized residues in biological samples is challenging. Herein, we introduce a fast and efficient method using tandem mass spectrometry (MS) to locate isomerized residues in peptides. Although MS spectra are useful for identifying peptides that contain an isomerized residue, they cannot reliably localize isomerization sites. We show that this limitation can be overcome by utilizing MS experiments to further evaluate each fragment ion from the MS stage. Comparison at the MS level, utilizing statistical analyses, reveals which MS fragments differ between samples and, therefore, must contain the isomerized sites. The approach is similar to previous work relying on ion mobility to discriminate MS product ions by collision cross-section. The MS approach can be implemented using either ion-trap or beam-type collisional activation and is compatible with the quantification of isomer mixtures when coupled to a calibration curve. The method can also be implemented in combination with liquid chromatography in a targeted approach. Enabling the identification and localization of isomerized residues in peptides with an MS-only methodology will expand accessibility to this important information.
已在从人和动物的组织或分泌物中提取的许多肽中鉴定出了异构氨基酸残基。这些异构残基可以通过影响生物活性或对衰老过程施加影响来发挥关键作用。异构化自发发生，不会导致质量转移。因此，在生物样品中鉴定和定位异构化残基具有挑战性。在此，我们介绍了一种使用串联质谱（MS）快速有效地定位肽中异构化残基的方法。尽管 MS 谱对于鉴定含有异构化残基的肽很有用，但它们无法可靠地定位异构化位点。我们表明，通过利用 MS 实验进一步评估 MS 阶段的每个片段离子，可以克服此限制。在 MS 水平上进行比较，并利用统计分析，可以揭示哪些 MS 片段在样品之间存在差异，因此必须包含异构化位点。该方法类似于先前依赖于离子淌度通过碰撞截面来区分 MS 产物离子的工作。可以使用离子阱或束型碰撞激活来实现 MS 方法，并且当与校准曲线结合使用时，该方法可以与异构混合物的定量兼容。该方法还可以与液相色谱结合以实现靶向方法。仅使用 MS 方法即可鉴定和定位肽中的异构化残基，将扩大获取此重要信息的途径。
