人类程序性细胞死亡蛋白 1 (hPD-1) 是免疫检查点通路中的重要受体。它在癌症治疗中发挥了重要作用。然而，并非所有患者都对 PD-1 抗体治疗产生积极反应，其潜在机制仍不清楚。 PD-1 是一种跨膜糖蛋白，据报道其胞外结构域 (ECD) 负责相互作用和信号转导。该结构域包含 4 个 N-糖基化位点和 25 个潜在的 O-糖基化位点，这暗示了糖基化的重要性。 hPD-1的结构已被深入研究，但该蛋白的糖基化，特别是每个糖基化位点上的聚糖尚未得到全面阐明。本研究分析了人胚肾293（HEK 293）和中国仓鼠卵巢（CHO）细胞表达的hPD-1 ECD；使用质谱法不仅对N-和O-糖基化位点进行了全面分析，还对这些位点上的聚糖进行了全面分析。此外，测试了hPD-1 ECD与不同抗hPD-1抗体的结合，发现N-聚糖的功能不同。所有这些聚糖信息都将有利于未来的 PD-1 研究。

Human programmed cell death protein 1 (hPD-1) is an essential receptor in the immune checkpoint pathway. It has played an important role in cancer therapy. However, not all patients respond positively to the PD-1 antibody treatment, and the underlying mechanism remains unknown. PD-1 is a transmembrane glycoprotein, and its extracellular domain (ECD) is reported to be responsible for interactions and signal transduction. This domain contains 4 N-glycosylation sites and 25 potential O-glycosylation sites, which implicates the importance of glycosylation. The structure of hPD-1 has been intensively studied, but the glycosylation of this protein, especially the glycan on each glycosylation site, has not been comprehensively illustrated. In this study, hPD-1 ECD expressed by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells was analyzed; not only N- and O-glycosylation sites but also the glycans on these sites were comprehensively analyzed using mass spectrometry. In addition, hPD-1 ECD binding to different anti-hPD-1 antibodies was tested, and N-glycans were found functioned differently. All of this glycan information will be beneficial for future PD-1 studies.