Routine o-glycan characterization in nutritional supplements -: a comparison of analytical methods for the monitoring of the bovine kappa-casein macropeptide glycosylation
Routine o-glycan characterization in nutritional supplements -: a comparison of analytical methods for the monitoring of the bovine kappa-casein macropeptide glycosylation
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DOI：
10.1016/s0021-9673(01)01176-1
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发表时间：
2001-09-21
影响因子：
4.1
通讯作者：
Veuthey, JL
中科院分区：
文献类型：
--
作者：
Tran, NT;Daali, Y;Veuthey, JL
采用毛细管等电聚焦（CIEF）、高效阴离子交换色谱耦合安培检测（HPAEC-PAD）和正相色谱与荧光检测等分析方法，对高o糖基化酪蛋白大肽（CGMP）进行了表征，并对两种不同制备工艺获得的CGMP批次进行了细微糖基化差异的检测。改进的两步CIEF可以监测糖肽的异质性和测定酸性糖型的等电点。优化了宽、窄两种pH范围两性电解质的混合物，以提高糖型的分辨率。用pl内标法测定不同CGMP糖型的pl值，发现其范围在3.08 ~ 3.58之间，表明其为酸性很强的糖肽。此外，通过适当的酸性水解CGMP，用HPAEC-PAD测定中性糖和氨基糖释放后的单糖组成。结果表明，第1批和第2批的组成相似，但第1批的单糖百分比高出3-4倍，特别是半乳糖和葡萄糖。这可能反映了第一批的乳糖含量较高。最后，通过自动肼解释放o -连接的低聚糖，并用敏感的标记试剂2-氨基苯甲酰胺衍生化。然后用正相高效液相色谱结合荧光检测对衍生物进行分析，并根据亲水性相互作用进行分离，从而对两种CGMP进行低聚糖定位。两种CGMP制剂具有几乎相同的o -聚糖群，但CGMP第1批比第2批糖基化程度更高。此外，分离的聚糖的大小，表示为葡萄糖单位的数量，使用部分水解葡聚糖的校准暂定。总之，电泳和色谱技术的结合在研究糖蛋白异质性和评估批间一致性方面是强有力的。(C) 2001 Elsevier Science B.V.版权所有
Analytical procedures, including capillary isoelectric focusing (CIEF), high-performance anion-exchange chromatography coupled to amperometric detection (HPAEC-PAD) and normal-phase chromatography with fluorescence detection are presented for the characterization of a highly O-glycosylated caseinomacropeptide (CGMP) and the detection of subtle glycosylation differences between CGMP Batches obtained with two different preparation procedures. Modified two-step CIEF allowed monitoring of glycopeptide heterogeneity and determination of the isoelectric points of acidic glycoforms. The mixture of wide and narrow pH range ampholytes was optimized to improve glycoform resolution. The pl of the different CGMP glycoforms was evaluated with pl internal standards and found to range between 3.08 and 3.58, which indicates a very acidic glycopeptide. Moreover, the monosaccharide composition was determined with HPAEC-PAD after neutral and amino sugars release by using adequate acidic hydrolysis of CGMP. Results indicated a similar composition for Batches I and II, but the monosaccharide percentages were 3-4 fold higher in Batch I, particularly for galactose and glucose. This likely reflects a higher content in lactose in the case of Batch I. Finally, O-linked oligosaccharides were released with an automated hydrazinolysis and derivatized with a sensitive labelling reagent, 2-aminobenzamide. The derivatives were then analyzed by normal-phase HPLC coupled with fluorescence detection, and separated on the basis of hydrophilic interaction, which allowed oligosaccharide mapping of the two CGMP. It appeared that the two CGMP preparations had an almost identical O-glycan population, but CGMP Batch I was more glycosylated than Batch II. Additionally, the sizes of the separated glycans, expressed as the number of glucose units, were tentatively assigned using calibration with a partial hydrolysate of dextran. In conclusion, a combination of electrophoretic and chromatographic techniques was found powerful in studying glycoprotein heterogeneity and assessing batch-to-batch consistency. (C) 2001 Elsevier Science B.V. All rights reserved.