Reiding Karli R, Blank Dennis, Kuijper Dennis M, Deelder André M, Wuhrer Manfred
Center for Proteomics and Metabolomics, Leiden University Medical Center , 2300 RC Leiden, The Netherlands.
Anal Chem. 2014 Jun 17;86(12):5784-93. doi: 10.1021/ac500335t. Epub 2014 May 28.
Protein glycosylation is an important post-translational modification associated, among others, with diseases and the efficacy of biopharmaceuticals. Matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS) can be performed to study glycosylation in a high-throughput manner, but is hampered by the instability and ionization bias experienced by sialylated glycan species. Stabilization and neutralization of these sialic acids can be achieved by permethylation or by specific carboxyl group derivatization with the possibility of discrimination between α2,3- and α2,6-linked sialic acids. However, these methods typically require relatively pure glycan samples, show sensitivity to side reactions, and need harsh conditions or long reaction times. We established a rapid, robust and linkage-specific high-throughput method for sialic acid stabilization and MALDI-TOF-MS analysis, to allow direct modification of impure glycan-containing mixtures such as PNGase F-released human plasma N-glycome. Using a combination of carboxylic acid activators in ethanol achieved near-complete ethyl esterification of α2,6-linked sialic acids and lactonization of α2,3-linked variants, in short time using mild conditions. Glycans were recovered by hydrophilic interaction liquid chromatography solid phase extraction and analyzed by MALDI-TOF-MS in reflectron positive mode with 2,5-dihydroxybenzoic acid as the matrix substance. Analysis of the human plasma N-glycome allowed high-throughput detection and relative quantitation of more than 100 distinct N-glycan compositions with varying sialic acid linkages.
蛋白质糖基化是一种重要的翻译后修饰，与疾病及生物制药的疗效等诸多方面相关。基质辅助激光解吸/电离（MALDI）飞行时间（TOF）质谱（MS）可用于高通量研究糖基化，但唾液酸化聚糖种类的不稳定性和电离偏倚限制了其应用。通过全甲基化或特定羧基衍生化可实现这些唾液酸的稳定化和中和，从而区分α2,3-和α2,6-连接的唾液酸。然而，这些方法通常需要相对纯净的聚糖样品，对副反应敏感，且需要苛刻条件或较长反应时间。我们建立了一种快速、稳健且具有连接特异性的高通量方法，用于唾液酸稳定化和MALDI-TOF-MS分析，可直接修饰含不纯聚糖的混合物，如PNGase F释放的人血浆N-糖组。在乙醇中使用羧酸活化剂的组合，在温和条件下短时间内实现了α2,6-连接唾液酸的近乎完全乙酯化和α2,3-连接变体的内酯化。通过亲水相互作用液相色谱固相萃取回收聚糖，并以2,5-二羟基苯甲酸为基质物质，在反射正离子模式下通过MALDI-TOF-MS进行分析。对人血浆N-糖组的分析实现了对100多种具有不同唾液酸连接的独特N-聚糖组成的高通量检测和相对定量。
