High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry.
High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry.
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DOI：
10.1021/ac1011377
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发表时间：
2010-08-01
影响因子：
7.4
通讯作者：
Van Cottt, Kevin E.
中科院分区：
文献类型：
--
作者：
Gil, Geun-Cheol;Iliff, Bryce;Cerny, Ron;Velander, William H.;Van Cottt, Kevin E.
重组治疗性糖蛋白的适当糖基化在生物制药工业中一直受到重视，因为碳水化合物成分会影响糖蛋白的安全性、有效性和一致性。可靠的定量方法是必不可少的，以确保其产品的一致性，关于糖基化，特别是唾液化。质谱法（MS）具有高分辨率、高灵敏度和结构鉴定能力，已成为分析多糖谱和结构的常用工具。然而，由于中性和酸性聚糖之间的电离效率不同，使用质谱法定量唾液化聚糖并不可靠。我们在这里报道，在温和的酸性条件下酰胺化可以用来中和酸性n -聚糖仍然附着在蛋白质。然后利用PNGase F将修饰后的n -聚糖从蛋白质中释放出来，并在还原端进行永久电荷标记，以避免在MS分析过程中发生任何修饰和金属加合物的形成。n -聚糖修饰、消化和脱盐步骤使用单锅方法进行，该方法可以在微离心管或96孔微滤板中进行，从而实现高通量聚糖分析。使用该方法，我们能够对重组人糖蛋白进行定量MALDI-TOF质谱分析，以确定聚焦化和唾液化的变化，这与正常相HPLC低聚糖定位方法非常一致。
Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method.
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影响因子：
7.4
作者：
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通讯作者：
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通讯作者：
Shiloach, Joseph