Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon–liquid chromatography–tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14⁺ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man_1-3GlcNAc₂Fuc_0–1, 22.1–30.8%), oligomannosidic (Man_5-9GlcNAc₂, 29.8–35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6–39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14⁺ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.

中文翻译：

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人类单核细胞到巨噬细胞转变的高分辨率纵向 N-和 O-糖谱分析。

蛋白质糖基化影响先天免疫细胞的发育和功能。与来自单核细胞前体群体的巨噬细胞成熟相关的糖表型和聚糖重塑仍未完全描述。在此，采用无标记多孔石墨化碳-液相色谱-串联质谱法 (PGC-LC-MS/MS) 以高分辨率分析与人类单核细胞到巨噬细胞转变相关的N - 和O -糖组。原代血液来源的 CD14 ⁺单核细胞在体外分化在没有强抗炎和促炎刺激的情况下，使用传统的 7 天粒细胞-巨噬细胞集落刺激因子分化方案进行纵向采样。通过光学显微镜和蛋白质组学监测的形态学和蛋白质表达验证了成熟过程。糖组学证明单核细胞和巨噬细胞显示出相似的N-糖组谱，主要包括少甘露糖苷 (Man _1-3 GlcNAc ₂ Fuc _0-1 , 22.1–30.8%)、寡甘露糖苷 (Man _5-9 GlcNAc ₂ , 29.8–35.7%) 和 α ,3/6-唾液酸化复合型N-具有可变核心岩藻糖基化的聚糖 (27.6–39.1%)。糖肽分析验证了这些聚糖与人类蛋白质的结合，而定量蛋白质组学监测了巨噬细胞分化过程中的糖酶表达水平。观察到显着的人际糖组变异，表明 CD14 ⁺单核细胞具有相当大的生理依赖性或可遗传的异质性。只有少数N-糖组变化与供体之间的单核细胞到巨噬细胞的转变相关，包括核心岩藻糖基化减少和甘露糖终止（少甘露糖苷型/寡甘露糖苷型）N 的表达降低巨噬细胞中的 -聚糖，而凝集素流式细胞术表明在成熟过程中发生了更显着的细胞表面聚糖重塑。异质性较低的富含核心 1 的O-糖组显示核心 2 型O-糖基化略有下降，但在巨噬细胞成熟时保持不变。这种支持正常单核细胞到巨噬细胞转变的高分辨率糖组图是迄今为止最详细的，有助于我们了解与两种重要的先天免疫细胞类型有关的分子构成，并为未来的糖免疫学研究提供重要参考。