靶向程序性细胞死亡1/程序性细胞死亡配体-1（PD-1/PD-L1）的免疫检查点抑制剂（ICIs）可显着延长晚期/转移性肺癌患者的生存期。然而，只有一小部分患者可以从 ICI 中受益，并且治疗过程的临床管理仍然具有挑战性。糖基化为增进我们对肿瘤免疫和免疫治疗的理解增加了一个新的维度。为了系统地表征抗 PD-1/PD-L1 免疫治疗相关的血清糖蛋白变化，我们在 ICI 治疗前和治疗期间收集了 12 名转移性肺鳞状细胞癌 (SCC) 和肺腺癌 (ADC) 患者的一系列血清样本，首先使用基于质谱的无标记定量方法进行分析。其次，在抗 PD-1/PD-L1 应答者和无应答者中进行分层分析，血清糖肽水平与治疗反应相关。此外，在一个独立的验证队列中，采用基于化学标记的大规模位点特异性分析策略来确认与抗 PD-1/PD-L1 治疗相关的 IgG N-糖基化的不寻常特征。无偏倚无标记定量糖蛋白组学揭示了 337 种定量糖肽中 27 种与抗 PD-1/PD-L1 治疗相关的血清水平变化。 IgG4对应的完整糖肽EEQFN 177STYR（H3N4）在抗PD-1/PD-L1治疗期间显着增加（FC=2.65，P=0.0083），并且在抗PD-1/PD-L1应答者中增加最高（FC=5.84，P=0.0190）。基于蛋白质纯化和化学标记的定量糖蛋白质组学证实了这一观察结果。此外，观察到两种完整糖肽（IgG4的EEQFN 177STYR（H3N4）、IgG3的EEQYN 227STFR（H3N4F1））与治疗反应之间存在明显的关联，这可能在癌症免疫治疗中发挥指导作用。我们的研究结果可能有益于未来的临床疾病管理。

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1/programmed cell death ligand-1 (PD-1/PD-L1) have significantly prolonged the survival of advanced/metastatic patients with lung cancer. However, only a small proportion of patients can benefit from ICIs, and clinical management of the treatment process remains challenging. Glycosylation has added a new dimension to advance our understanding of tumor immunity and immunotherapy. To systematically characterize anti-PD-1/PD-L1 immunotherapy-related changes in serum glycoproteins, a series of serum samples from 12 patients with metastatic lung squamous cell carcinoma (SCC) and lung adenocarcinoma (ADC), collected before and during ICIs treatment, are firstly analyzed with mass-spectrometry-based label-free quantification method. Second, a stratification analysis is performed among anti-PD-1/PD-L1 responders and non-responders, with serum levels of glycopeptides correlated with treatment response. In addition, in an independent validation cohort, a large-scale site-specific profiling strategy based on chemical labeling is employed to confirm the unusual characteristics of IgG N-glycosylation associated with anti-PD-1/PD-L1 treatment. Unbiased label-free quantitative glycoproteomics reveals serum levels' alterations related to anti-PD-1/PD-L1 treatment in 27 out of 337 quantified glycopeptides. The intact glycopeptide EEQFN 177STYR (H3N4) corresponding to IgG4 is significantly increased during anti-PD-1/PD-L1 treatment (FC=2.65, P=0.0083) and has the highest increase in anti-PD-1/PD-L1 responders (FC=5.84, P=0.0190). Quantitative glycoproteomics based on protein purification and chemical labeling confirms this observation. Furthermore, obvious associations between the two intact glycopeptides (EEQFN 177STYR (H3N4) of IgG4, EEQYN 227STFR (H3N4F1) of IgG3) and response to treatment are observed, which may play a guiding role in cancer immunotherapy. Our findings could benefit future clinical disease management.