An understanding of why hydrophilic interaction liquid chromatography gives a higher resolution for glycans than for glycoproteins would facilitate column improvements. Separations of the glycoforms of ribonuclease B compared to its released glycans were studied using a commercial hydrophilic interaction liquid chromatography column. The findings were used to devise a new hydrophilic interaction liquid chromatography column. For the commercial column, chromatograms and van Deemter plots showed that selectivity and efficiency are comparable factors in the higher resolution of the released glycans. The higher selectivity for the released glycans was associated with more water molecules displaced per added mannose. To investigate why, three‐dimensional structures of the glycoprotein and the glycan were computed under chromatographic conditions. These showed that hydrogen bonding within the free glycan makes its topology more planar, which would increase contact with the bonded phase. The protein sterically blocks the hydrogen bonding. The more globular‐shaped glycan of the glycoprotein suggests that a thicker bonded phase might improve selectivity. This was tested by making a column with a copolymer bonded phase. The results confirmed that selectivity is increased. The findings are possibly broadly relevant to glycoprotein analysis since the structural motif involved in internal hydrogen bonding is common to N‐linked glycans of human glycoproteins.

中文翻译：

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蛋白质诱导的聚糖构象变化降低了亲水相互作用液相色谱中糖蛋白的分辨率

了解为什么亲水相互作用液相色谱对聚糖的分离度高于糖蛋白的分离度，将有助于色谱柱的改进。使用商业亲水相互作用液相色谱柱研究了核糖核酸酶 B 的糖型与其释放的聚糖的分离。这些发现被用于设计一种新的亲水相互作用液相色谱柱。对于商业色谱柱，色谱图和 van Deemter 图表明，选择性和柱效是释放聚糖更高分离度的可比因素。释放聚糖的更高选择性与每添加甘露糖置换更多的水分子有关。为了研究原因，在色谱条件下计算了糖蛋白和聚糖的三维结构。这些表明游离聚糖内的氢键使其拓扑结构更加平坦，这将增加与键合相的接触。蛋白质在空间上阻断氢键。糖蛋白的球状聚糖越多表明键合相越厚可能会提高选择性。这是通过制造具有共聚物键合相的色谱柱来测试的。结果证实选择性增加。这些发现可能与糖蛋白分析广泛相关，因为参与内部氢键的结构基序对于人类糖蛋白的 N 连接聚糖是常见的。糖蛋白的球状聚糖越多表明键合相越厚可能会提高选择性。这是通过制造具有共聚物键合相的色谱柱来测试的。结果证实选择性增加。这些发现可能与糖蛋白分析广泛相关，因为参与内部氢键的结构基序对于人类糖蛋白的 N 连接聚糖是常见的。糖蛋白的球状聚糖越多表明键合相越厚可能会提高选择性。这是通过制造具有共聚物键合相的色谱柱来测试的。结果证实选择性增加。这些发现可能与糖蛋白分析广泛相关，因为参与内部氢键的结构基序对于人类糖蛋白的 N 连接聚糖是常见的。