Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana, USA.
Genentech, Inc. One DNA Way, South San Francisco, California, USA.
J Sep Sci. 2021 Apr;44(8):1581-1591. doi: 10.1002/jssc.202001242. Epub 2021 Mar 31.
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.
理解为什么亲水相互作用液相色谱法对聚糖的分辨率比对糖蛋白的分辨率更高，将有助于改进柱。使用商业亲水相互作用液相色谱柱研究了核糖核酸酶 B 的糖型与释放糖的分离。研究结果用于设计一种新的亲水相互作用液相色谱柱。对于商业柱，色谱图和范德姆特图表明，选择性和效率是释放糖高分辨率的可比因素。释放糖的更高选择性与每添加一个甘露糖就会取代更多的水分子有关。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。这些结果表明，游离聚糖内的氢键使其拓扑结构更具平面性，这将增加与键合相的接触。蛋白质在空间上阻止了氢键的形成。糖蛋白的球形聚糖表明，更厚的键合相可能会提高选择性。这通过使用具有共聚键合相的色谱柱进行了测试。结果证实选择性得到了提高。由于涉及内部氢键的结构基序在人糖蛋白的 N 连接聚糖中很常见，因此这些发现可能广泛适用于糖蛋白分析。
