使用基质辅助激光解吸电离质谱技术对神经退行性疾病进行脑脊液N-糖蛋白质组学研究

CSF N-Glycoproteomics Using MALDI MS Techniques in Neurodegenerative Diseases.

作者信息

Messina Angela, Palmigiano Angelo, Bua Rosaria Ornella, Romeo Donata Agata, Barone Rita, Sturiale Luisa, Zappia Mario, Garozzo Domenico

机构信息

CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania, Catania, Italy.

Child Neurology and Psychiatry Unit, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy.

出版信息

Methods Mol Biol. 2019;2044:255-272. doi: 10.1007/978-1-4939-9706-0_16.

DOI:10.1007/978-1-4939-9706-0_16

PMID:31432418

Abstract

CSF diagnostics has proved to be a formidable testing ground for N-glycoproteomic analysis of neurological diseases. To characterize specific N-glycan profiles of CSF in early and advanced phases of Alzheimer's disease, as well as in lysosomal storage disorders such as Tay-Sachs disease, we set up in our lab a robust and feasible protocol by coupling bioanalytical methods and mass spectrometry analysis.Starting from a few microliters of CSF, after protein denaturation, reduction, and alkylation, N-glycans are released from glycoproteins using the peptide-N-glycosidase F (PNGase F) and purified. The analysis of permethylated N-glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS allowed us to identify specific glyco-structures and also to distinguish between isobaric N-glycans.

摘要

脑脊液诊断已被证明是神经疾病N-糖蛋白质组分析的一个强大试验场。为了表征阿尔茨海默病早期和晚期以及溶酶体贮积症（如泰-萨克斯病）中脑脊液的特定N-聚糖谱，我们在实验室中通过结合生物分析方法和质谱分析建立了一个稳健且可行的方案。从几微升脑脊液开始，经过蛋白质变性、还原和烷基化后，使用肽-N-糖苷酶F（PNGase F）从糖蛋白中释放N-聚糖并进行纯化。通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和MALDI-TOF MS/MS对全甲基化N-聚糖进行分析，使我们能够识别特定的糖结构，并区分等压N-聚糖。