Utilization of Liquid Chromatography–Mass Spectrometry and High-Resolution Ion Mobility–Mass Spectrometry to Characterize Therapeutically Relevant Peptides with Asparagine Deamidation and Isoaspartate

IF 6.7 1区化学 Q1 CHEMISTRY, ANALYTICAL

Analytical Chemistry Pub Date : 2024-12-23 DOI:10.1021/acs.analchem.4c05246

Andrew B. Dykstra, Thomas G. Lubinsky, Heidi Vitrac, Iain D. G. Campuzano, Pavel V. Bondarenko, Ashli R. Simone

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Abstract

Rapid identification of asparagine (Asn) deamidation and isoaspartate (isoAsp) in proteins remains a challenging analytical task during the development of biological therapeutics. For this study, 46 therapeutically relevant peptides corresponding to 13 peptide families (13 unmodified peptides and 33 modified peptides) were obtained; modified peptides included Asn deamidation and isoAsp. The peptide families were characterized by three methods: reversed-phase ultrahigh performance liquid chromatography–mass spectrometry (RP-UHPLC-MS); flow injection analysis high-resolution ion mobility–mass spectrometry (FIA-HRIM-MS); and shortened gradient RP-UHPLC-HRIM-MS. UHPLC-MS data acquisition was 2 h per injection, in contrast to high-throughput 1 min data acquisition of the FIA-HRIM-MS technique. A rapid 2D peptide map has been demonstrated by combining shortened gradient RP-UHPLC with HRIM, to optimize the resolution of the Asn-, Asp-, and isoAsp-containing peptides, increasing the likelihood of detecting peptides containing these quality attributes with expedited data acquisition. Additionally, this paper provides an ion mobility calibration data set for therapeutically relevant peptides (unmodified and modified) over an ion-neutral collisional cross-section range of 300–800 Å².

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利用液相色谱-质谱法和高分辨率离子迁移-质谱法表征天冬酰胺脱酰胺和异天冬氨酸治疗相关肽

在生物治疗学的发展过程中，快速鉴定蛋白质中的天冬酰胺（Asn）脱酰胺和异天冬氨酸（isoAsp）仍然是一个具有挑战性的分析任务。本研究共获得了13个肽家族（13个未修饰肽和33个修饰肽）的46个治疗相关肽；修饰肽包括Asn脱酰胺和isoAsp。采用反相超高效液相色谱-质谱法（RP-UHPLC-MS）对多肽家族进行了表征；流动注射分析高分辨率离子迁移质谱法（FIA-HRIM-MS）；缩短梯度RP-UHPLC-HRIM-MS。UHPLC-MS每次注射的数据采集时间为2小时，而FIA-HRIM-MS技术的高通量数据采集时间为1分钟。通过将缩短梯度的RP-UHPLC与hrm相结合，建立了快速的2D肽图，以优化含有Asn-、Asp-和isoasp -的肽的分辨率，增加了检测含有这些质量属性的肽的可能性，加快了数据采集。此外，本文还提供了治疗相关肽（未修饰和修饰）在300-800离子中性碰撞截面范围内的离子迁移率校准数据集Å2。

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来源期刊

Analytical Chemistry 化学-分析化学

CiteScore

12.10

自引率

12.20%

发文量

1949

审稿时长

1.4 months

期刊介绍： Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.