Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, USA.
Center for Human Health and the Environment, North Carolina State University, Raleigh, NC, 27695, USA.
Anal Bioanal Chem. 2022 Jan;414(1):623-637. doi: 10.1007/s00216-021-03570-7. Epub 2021 Aug 4.
Glycosylation is a ubiquitous co- and post-translational modification involved in the sorting, folding, and trafficking of proteins in biological systems; in humans, >50% of gene products are glycosylated with the cellular machinery of glycosylation compromising ~2% of the genome. Perturbations in glycosylation have been implicated in a variety of diseases including neurodegenerative diseases and certain types of cancer. However, understanding the relationship between a glycan and its biological role is often difficult due to the numerous glycan isomers that exist. To address this challenge, nanoflow liquid chromatography, ion mobility spectrometry, and mass spectrometry (nLC-IMS-MS) were combined with the Individuality Normalization when Labeling with the Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy to study a series of glycan standards and those enzymatically released from the glycoproteins horseradish peroxidase, fetuin, and pooled human plasma. The combination of IMS and the natural (NAT) and stable-isotope label (SIL) in the INLIGHT™ strategy provided additional confidence for each glycan identification due to the mobility aligned NAT- and SIL-labeled glycans and further capabilities for isomer examinations. Additionally, molecular trend lines based on the IMS and MS dimensions were investigated for the INLIGHT™ derivatized glycans, facilitating rapid identification of putative glycans in complex biological samples.
糖基化是一种普遍存在的共翻译和翻译后修饰，参与生物系统中蛋白质的分拣、折叠和运输；在人类中，>50%的基因产物都被糖基化，糖基化的细胞机制涉及到约 2%的基因组。糖基化的改变与多种疾病有关，包括神经退行性疾病和某些类型的癌症。然而，由于存在大量的聚糖异构体，了解聚糖与其生物学功能之间的关系常常很困难。为了解决这一挑战，纳流液相色谱、离子淌度质谱和质谱（nLC-IMS-MS）与同位素糖基化酰肼标签的个体归一化标记（INLIGHT™）策略相结合，用于研究一系列聚糖标准品和从辣根过氧化物酶、胎球蛋白和混合人血浆中酶解释放的聚糖。由于 INLIGHT™策略中的 IMS 以及天然（NAT）和稳定同位素标记（SIL）的结合，提供了每个聚糖鉴定的额外信心，因为迁移对齐的 NAT 和 SIL 标记的聚糖，以及进一步的异构体检查能力。此外，还研究了基于 IMS 和 MS 维度的分子趋势线，用于 INLIGHT™衍生的聚糖，促进了在复杂生物样本中快速鉴定可能的聚糖。
