Deamidation is a spontaneous degenerative protein modification (DPM) that disrupts the structure and function of both endogenous proteins and various therapeutic agents. While deamidation has long been recognized as a critical event in human aging and multiple degenerative diseases, research progress in this field has been restricted by the technical challenges associated with studying this DPM in complex biological samples. Asparagine (Asn) deamidation generates L-aspartic acid (L-Asp), D-aspartic acid (D-Asp), L-isoaspartic acid (L-isoAsp) or D-isoaspartic acid (D-isoAsp) residues at the same position of Asn in the affected protein, but each of these amino acids displays similar hydrophobicity and cannot be effectively separated by reverse phase liquid chromatography. The Asp and isoAsp isoforms are also difficult to resolve using mass spectrometry since they have the same mass and fragmentation pattern in MS/MS. Moreover, the ¹³C peaks of the amidated peptide are often misassigned as monoisotopic peaks of the corresponding deamidated peptides in protein database searches. Furthermore, typical protein isolation and proteomic sample preparation methods induce artificial deamidation that cannot be distinguished from the physiological forms. To better understand the role of deamidation in biological aging and degenerative pathologies, new technologies are now being developed to address these analytical challenges, including mixed mode electrostatic-interaction modified hydrophilic interaction liquid chromatography (emHILIC). When coupled to high resolution, high accuracy tandem mass spectrometry this technology enables unprecedented, proteome-wide study of the ‘deamidome’ of complex samples. The current article therefore reviews recent advances in sample preparation methods, emHILIC-MS/MS technology, and MS instrumentation / data processing approaches to achieving accurate and reliable characterization of protein deamidation in complex biological and clinical samples.

脱酰胺是一种自发的退行性蛋白质修饰 (DPM)，它会破坏内源性蛋白质和各种治疗剂的结构和功能。虽然脱酰胺长期以来被认为是人类衰老和多种退行性疾病的关键事件，但该领域的研究进展受到与在复杂生物样品中研究这种 DPM 相关的技术挑战的限制。天冬酰胺 (Asn) 脱酰胺在同一位置生成 L-天冬氨酸 (L-Asp)、D-天冬氨酸 (D-Asp)、L-异天冬氨酸 (L-isoAsp) 或 D-异天冬氨酸 (D-isoAsp) 残基受影响的蛋白质中的 Asn，但这些氨基酸中的每一个都显示出相似的疏水性，并且不能通过反相液相色谱法有效分离。Asp 和 isoAsp 异构体也难以使用质谱分析，因为它们在 MS/MS 中具有相同的质量和碎裂模式。此外，¹³在蛋白质数据库搜索中，酰胺化肽的 C 峰经常被错误指定为相应脱酰胺化肽的单同位素峰。此外，典型的蛋白质分离和蛋白质组学样品制备方法会导致无法与生理形式区分开来的人工脱酰胺作用。为了更好地了解脱酰胺在生物老化和退行性病变中的作用，目前正在开发新技术来应对这些分析挑战，包括混合模式静电相互作用改性亲水相互作用液相色谱法 (emHILIC)。当与高分辨率、高精度串联质谱仪结合使用时，该技术可以对复杂样品的“脱酰胺组”进行前所未有的全蛋白质组研究。因此，本文回顾了样品制备方法的最新进展，