Young Clifford, Condina Mark R, Briggs Matthew T, Moh Edward S X, Kaur Gurjeet, Oehler Martin K, Hoffmann Peter
Future Industries Institute, University of South Australia, Adelaide, SA, Australia.
Department of Molecular Sciences, Macquarie University, Sydney, NSW, Australia.
Front Chem. 2021 Jun 11;9:653959. doi: 10.3389/fchem.2021.653959. eCollection 2021.
Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released glycans from a glycoprotein standard mixture, the majority of glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). glycans released from complex samples were also examined by PGC LC-MS. A total of 120 glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of glycans.
蛋白质糖基化是一种常见的翻译后修饰，可调节免疫反应和蛋白质运输等生物过程。糖基化谱的改变与癌症和炎症性疾病相关，也会影响治疗性单克隆抗体的疗效。连接聚糖由连接到天冬酰胺残基上的寡糖组成，由于存在多种异构结构，酶促释放的连接聚糖在分析上具有挑战性。液相色谱-质谱联用（LC-MS）是一种常用的聚糖定量分析技术，可用于聚糖的分离和表征。尽管许多反相和正相固定相已用于聚糖的分离，但多孔石墨化碳（PGC）色谱因其更高的分离能力而备受青睐，但难以以稳健且可重复的方式实施。在此，我们展示了一根15厘米长的熔融石英毛细管（内径75微米，外径360微米）的分析特性，该毛细管内部填充了Hypercarb PGC（3微米），并与安捷伦6550 Q-TOF质谱仪联用，用于在正离子模式下进行聚糖分析。在对糖蛋白标准混合物释放的聚糖进行的重复性和中间精密度测量中，大多数聚糖的保留时间变异系数较低（≤4.2%），峰面积变异系数较低（≤14.4%）。还通过PGC LC-MS对复杂样品释放的聚糖进行了检测。从福尔马林固定石蜡包埋的卵巢癌组织切片中总共获得了120种聚糖结构和组成异构体。最后，对早期和晚期福尔马林固定石蜡包埋的卵巢癌组织进行比较，发现一种岩藻糖基化双天线复合聚糖的α2,3-和α2,6-唾液酸连接存在定性变化。虽然α2,3-连接在晚期卵巢癌中占主导地位，但交替的α2,6-连接在早期卵巢癌中更为普遍。本研究确立了自制PGC柱在聚糖稳健且可重复的LC-MS分析中的实用性。
