Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague 2, Czech Republic.
Anal Bioanal Chem. 2024 Mar;416(8):1867-1881. doi: 10.1007/s00216-024-05187-y. Epub 2024 Feb 13.
The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.
样品制备步骤在糖蛋白质组学分析中至关重要。一种有效的糖蛋白样品制备方法涉及使用亲水相互作用色谱（HILIC）模式下的极性固定相通过固相萃取（SPE）来富集糖肽。本工作旨在展示不同实验条件如何影响氨基丙基改性 SPE 柱上人免疫球蛋白 G（IgG）的糖肽的富集效率。不同洗脱溶剂（乙腈、甲醇和异丙醇）的组成，以及不同浓度的洗脱溶剂酸化剂（甲酸和乙酸），以及用于调节和洗涤溶剂的不同浓度的乙腈（65%、75%和 85%乙腈）进行了测试，以观察它们对糖肽富集过程的影响。异丙醇在富集糖肽方面效果较差，而乙腈则是最有效的，甲醇则介于两者之间。洗脱溶剂中较高浓度的甲酸削弱了离子相互作用，特别是与唾液酸化的糖肽。用乙酸替代甲酸导致更多的糖肽更早洗脱。调节和洗涤溶液中的乙腈浓度起着关键作用；在 65%乙腈时，糖肽未保留在 SPE 柱上，而在流穿部分中检测到。最终，证明该富集方法适用于人血浆样品，导致非糖基化肽的丰度显著降低。据我们所知，这是首次在 HILIC 模式下使用氨基丙基改性 SPE 柱系统研究流动相对糖肽富集的影响。这项研究表明，实验条件的微小变化会对 SPE-HILIC 糖肽富集产生重大影响，而这些变化在文献中尚未考虑到。因此，必须仔细优化这些条件，以增强糖蛋白质组学分析的特异性和选择性，确保准确可靠的定量。
