蛋白质磷酸化是最常见和最重要的翻译后修饰之一，调节几乎所有生命过程。特别是，蛋白质磷酸化调节肿瘤、神经退行性疾病和糖尿病等重大疾病的发展。例如，Tau蛋白过度磷酸化会导致神经原纤维缠结，导致阿尔茨海默病。因此，必须开发大规模鉴定蛋白质磷酸化的方法。高效富集方法和生物质谱技术的快速发展使得低丰度蛋白质O-磷酸化修饰的大规模鉴定成为可能，从而可以更深入地研究其生物学功能。组氨酸、精氨酸和赖氨酸侧链氨基上发生的 N-磷酸化修饰最近受到越来越多的关注。例如，原核生物中组氨酸磷酸化的生物学功能已得到充分研究；这种类型的修饰调节信号转导和糖代谢。已使用各种生物学方法成功鉴定了两种哺乳动物 pHis 激酶（NME1 和 NME2）和三种 pHis 磷酸酶（PHPT1、LHPP 和 PGAM5）。 N-磷酸化参与多种生物过程，其功能不可忽视。然而，由于P-N键的化学稳定性差，N-磷酸化在酸性和热条件下不稳定。遗憾的是，目前依赖酸性条件的O-磷酸化富集方法并不适合N-磷酸化富集，导致蛋白质N-磷酸化的大规模鉴定严重滞后。富集方法的缺乏也严重阻碍了N-磷酸化生物学功能的研究。因此，开发针对蛋白质N-磷酸化的高效富集方法刻不容缓。 N-磷酸化蛋白质组富集方法的研究有限，阻碍了功能研究。因此，总结这些方法对于促进进一步的功能研究是必要的。本文介绍了蛋白质N-磷酸化的结构特点和报道的生物学功能，回顾了过去二十年发展起来的蛋白质N-磷酸化修饰富集方法，并分析了每种方法的优缺点。在本研究中，详细描述了基于抗体的方法和非抗体依赖性方法。由于组氨酸分子结构的稳定性，抗体方法目前仅限于组氨酸磷酸化富集研究。未来的研究将集中在新的富集配体的开发上。此外，配体的研究将促进其他非常规磷酸化靶点的研究，例如二酰基磷酸（pAsp、pGlu）和S-磷酸（pCys）。综上所述，本文对N-磷酸化富集方法的历史和发展方向进行了详细分析。

Protein phosphorylation is one of the most common and important post-translational modifications that regulates almost all life processes. In particular, protein phosphorylation regulates the development of major diseases such as tumors, neurodegenerative diseases, and diabetes. For example, excessive phosphorylation of Tau protein can cause neurofibrillary tangles, leading to Alzheimer's disease. Therefore, large-scale methods for identifying protein phosphorylation must be developed. Rapid developmentin efficient enrichment methods and biological mass spectrometry technologies have enabled the large-scale identification of low-abundance protein O-phosphorylation modifications in, allowing for a more thorough study of their biological functions. The N-phosphorylation modifications that occur on the side-chain amino groups of histidine, arginine, and lysine have recently received increased attention. For example, the biological function of histidine phosphorylation in prokaryotes has been well studied; this type of modification regulates signal transduction and sugar metabolism. Two mammalian pHis kinases (NME1 and NME2) and three pHis phosphatases (PHPT1, LHPP, and PGAM5) have been successfully identified using various biological methods. N-Phosphorylation is involved in multiple biological processes, and its functions cannot be ignored. However, N-phosphorylation is unstable under acidic and thermal conditions owing to the poor chemical stability of the P-N bond. Unfortunately, the current O-phosphorylation enrichment method, which relies on acidic conditions, is unsuitable for N-phosphorylation enrichment, resulting in a serious lag in the large-scale identification of protein N-phosphorylation. The lack of enrichment methods has also seriously hindered studies on the biological functions of N-phosphorylation. Therefore, the development of efficient enrichment methods that target protein N-phosphorylation is an urgent undertaking. Research on N-phosphorylation proteome enrichment methods is limited, hindering functional research. Thus, summarizing such methods is necessary to promote further functional research. This article introduces the structural characteristics and reported biological functions of protein N-phosphorylation, reviews the protein N-phosphorylation modification enrichment methods developed over the past two decades, and analyzes the advantages and disadvantages of each method. In this study, both antibody-based and nonantibody-dependent methods are described in detail. Owing to the stability of the molecular structure of histidine, the antibody method is currently limited to histidine phosphorylation enrichment research. Future studies will focus on the development of new enrichment ligands. Moreover, research on ligands will promote studies on other nonconventional phosphorylation targets, such as two acyl-phosphates (pAsp, pGlu) and S-phosphate (pCys). In summary, this review provides a detailed analysis of the history and development directions of N-phosphorylation enrichment methods.