<br>使用 193 nm 紫外光解离质谱法区分天冬氨酸和异天冬氨酸,Analytical Chemistry

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使用 193 nm 紫外光解离质谱法区分天冬氨酸和异天冬氨酸
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-07-17 , DOI: 10.1021/acs.analchem.3c02025
Aarti Bashyal ₁ , John O Hui ₂ , Tawnya Flick ₃ , Andrew B Dykstra ₃ , Qingchun Zhang ₃ , Iain D G Campuzano ₂ , Jennifer S Brodbelt ₁

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天冬氨酸 (Asp) 自发转化为异天冬氨酸 ( iso Asp) 是一种普遍存在的修饰，会影响蛋白质的结构和功能。 Asp 的这种修饰会影响生物治疗药物的稳定性，并与神经退行性疾病的发展有关。我们探索了使用 193 nm 紫外光解离 (UVPD) 来区分质子化和去质子化肽中的天冬氨酸和异天冬氨酸。 UVPD 独特产生的几种碎片离子的相对丰度差异用于区分含有 Asp 或iso Asp 的异构肽标准品。这些碎片离子是由于 Asp/ iso Asp 残基的 N 端键断裂以及 Asp/ iso Asp 的侧链损失或y离子的 COOH、CO ₂ 、CO 或 H ₂ O 损失而产生的。。使用 UVPD 对含 Asp 的胰蛋白酶肽进行裂解，产生更多增强的w / w + 1/ y – 1/ x离子，而含iso Asp 的肽则产生更多增强的y – 18/ y – 45/ y – 46 离子。 UVPD 还用于从单克隆抗体的胰蛋白酶消化物中鉴定异构化肽。此外，质子化非胰蛋白酶肽的 UVPD 导致异天冬氨酸 N 端和 C 端的y离子更多增强，以及用于鉴定异天冬氨酸肽的b / y离子比率差异。

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Differentiation of Aspartic and Isoaspartic Acid Using 193 nm Ultraviolet Photodissociation Mass Spectrometry

Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid (isoAsp) is a ubiquitous modification that influences the structure and function of proteins. This modification of Asp impacts the stability of biotherapeutics and has been linked to the development of neurodegenerative diseases. We explored the use of 193 nm ultraviolet photodissociation (UVPD) to distinguish Asp and isoAsp in the protonated and deprotonated peptides. The differences in the relative abundances of several fragment ions uniquely generated by UVPD were used to differentiate isomeric peptide standards containing Asp or isoAsp. These fragment ions result from the cleavage of bonds N-terminal to Asp/isoAsp residues in addition to the side-chain losses from Asp/isoAsp or the losses of COOH, CO₂, CO, or H₂O from y-ions. Fragmentation of Asp-containing tryptic peptides using UVPD resulted in more enhanced w/w + 1/y – 1/x ions, while isoAsp-containing peptides yielded more enhanced y – 18/y – 45/y – 46 ions. UVPD was also used to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Moreover, UVPD of a protonated nontryptic peptide resulted in more enhanced y ions N- and C-terminal to isoAsp and differences in b/y ion ratios that were used to identify the isoAsp peptide.

更新日期：2023-07-17

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