Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
Anal Chem. 2017 May 16;89(10):5364-5372. doi: 10.1021/acs.analchem.7b00062. Epub 2017 Apr 26.
Exosomes are extracellular nanosized vesicles with lipid bilayers encapsulating nucleic acids and proteins, both with and without glycosylation. While exosomal nucleic acids and proteins have previously been explored to identify cancer biomarkers with some promising results, little information has been available concerning their glycoconjugate content. Exosomes were isolated from normal urine samples through multistep differential centrifugation. The isolated exosomes have an average size of 146 nm and a spherical shape, as determined by dynamic light scattering and transmission electron microscopy, respectively. N-Glycans were enzymatically released from the isolated vesicles. After being reduced and permethylated, N-glycans were measured by MALDI mass spectrometry. Paucimannosidic, high-mannose, and complex type glycans were identified and their relative abundances were determined. Some detailed structures of these glycans were revealed through liquid chromatography/tandem mass spectrometry (LC/MS-MS). The reduced N-glycans, without being permethylated, were also separated and analyzed by LC/MS-MS, and their structures were further detailed through isomeric separation on porous graphitized carbon (PGC) packed in long capillaries. Using microfractionation before LC/MS-MS, minor multiantennary N-glycans were preconcentrated as based on hydrophobicity or charge. Preconcentration of the reduced and permethylated glycans on a C18 cartridge revealed numerous large glycans, whereas fractionation of the reduced N-glycans by ion-exchange cartridges facilitated detection of sulfated glycans. After removing N-glycans from the original sample aliquot, O-glycans were chemically released from urinary exosomes and profiled, revealing some unusual structures.
外泌体是带有双层脂膜的纳米级细胞外囊泡，其中包含核酸和蛋白质，这些物质既有糖基化的也有非糖基化的。虽然已经有研究探索了外泌体中的核酸和蛋白质，以寻找有前景的癌症生物标志物，但关于其糖缀合物含量的信息却很少。通过多步差速离心从正常尿液样本中分离出外泌体。通过动态光散射和透射电子显微镜分别确定分离出的外泌体的平均大小为 146nm 和球形。从分离的囊泡中酶解释放 N-糖链。还原和全甲基化后，通过 MALDI 质谱法测量 N-糖链。鉴定了 pauci-mannosidic、高甘露糖和复合型糖，并确定了它们的相对丰度。通过液相色谱/串联质谱（LC/MS-MS）进一步揭示了这些糖的一些详细结构。未经全甲基化还原的 N-糖链也通过 LC/MS-MS 进行分离和分析，并通过在长毛细管中填充多孔石墨化碳（PGC）进行异构体分离进一步详细说明其结构。在 LC/MS-MS 之前使用微分级，基于疏水性或电荷预先浓缩具有多个天线的低聚糖。将还原和全甲基化的糖预浓缩在 C18 小柱上，揭示了许多大的糖，而通过离子交换小柱对还原的 N-糖进行分级分离则有助于检测硫酸化糖。从原始样品等分试样中去除 N-糖后，通过化学方法从尿外泌体中释放 O-糖并进行分析，揭示了一些不寻常的结构。
