Centre for Analytical Sciences Amsterdam, Amsterdam, the Netherlands; Division of BioAnalytical Chemistry, Amsterdam Institute for Molecular and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands; van 't Hoff Institute for Molecular Science, University of Amsterdam, Amsterdam, the Netherlands.
DSM Biotechnology Center, Analysis Department, Delft, the Netherlands.
Anal Chim Acta. 2020 May 1;1109:69-77. doi: 10.1016/j.aca.2020.02.042. Epub 2020 Feb 28.
Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches.
许多工业酶由于同时存在翻译后修饰而表现出宏观和微观异质性。由此产生的蛋白质异构体可能具有不同的活性和稳定性，因此，研究其分布对于酶产品的开发和监测具有重要意义。蛋白质糖基化可能起着关键作用，因为它可以影响酶的表达、物理和生化性质。我们报告了使用亲水相互作用液相色谱-质谱法（HILIC-MS）来分析高甘露糖型 N-糖基化蛋白质的完整糖型分布，以工业生产的用于食品工业的真菌脂肪酶为例。我们将这些结果与传统的反相液相色谱-质谱（RPLC-MS）和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行了比较。HILIC 在解析脂肪酶异质性方面表现出色，有助于 N-糖型和序列变体的质量分配。为了了解 HILIC 提供的糖型选择性，我们从脂肪酶的四个主要 HILIC 洗脱带中取出部分，并进行 SDS-PAGE 和自上而下的蛋白质组学分析。这些分析使我们能够鉴定每个馏分中存在的最丰富的糖基化位点，并证实了 HILIC 基于占据的糖基化位点数量来分离蛋白质糖型的能力。与 RPLC-MS 相比，HILIC-MS 减少了进入质谱仪的样品复杂性，有助于分配糖型和序列变体的质量，并增加了检测到的糖型数量（69 种更多的蛋白质异构体，增加了 177%）。HILIC-MS 方法需要相对较短的分析时间（<30 分钟），在此期间可区分 100 多种糖型。我们建议 HILIC(-MS)可以成为一种有价值的工具，用于表征旨在指导蛋白质糖型表达的生物工程过程，以及检查产品批次的一致性。
