Department of Chemistry and Biochemistry, Texas Tech University, United States.
Department of Chemistry and Biochemistry, Texas Tech University, United States.
J Chromatogr A. 2023 Sep 13;1706:464237. doi: 10.1016/j.chroma.2023.464237. Epub 2023 Jul 22.
Retention time is one of the most important parameters that has been widely used to demonstrate the separation results obtained from liquid chromatography (LC) platforms. However, retention time can shift when samples are tested with different instruments and laboratories, which hinders the identification process of analytes when comparing data collected from different LC systems. To address this problem, hydrophobicity index was introduced for retention time normalization of the glycopeptides separated by reversed-phase LC (RPLC). Tandem MS was used for the detection and identification of glycopeptides. In addition, the influence of different types of glycans on the hydrophobicity of peptide backbones was studied by comparing the retention time of glycopeptides with their non-glycosylated counterparts. The hydrophobicity of tryptic digested glycopeptides derived from model glycoproteins, including bovine fetuin, α1-acid glycoprotein, and haptoglobin from human plasma, were evaluated based on the hydrophobicity index of the standard peptides from a peptide retention time calibration mixture. The reduction of hydrophobicity of multiple peptide backbones was observed due to the hydrophilic glycan structures. By comparing the hydrophobicity index of glycopeptides collected from different time and instruments, the day-to-day and lab-to-lab comparisons suggested high reliability and reproducibility of this approach. The RSD% of hydrophobicity index from inter-lab experiments was 1.2%, while the RSD% of retention time was 5.1%. Then, the applications of this method were demonstrated on complex glycopeptide samples extracted from human blood serum. The hydrophobicity index can be applied to address the retention time shift when using different instruments, thereby boosting confidence of the characterization of glycopeptides.
保留时间是液相色谱（LC）平台中广泛用于展示分离结果的最重要参数之一。然而，当样品在不同的仪器和实验室中进行测试时，保留时间会发生偏移，这在比较来自不同 LC 系统的数据时会阻碍分析物的鉴定过程。为了解决这个问题，引入了疏水性指数来对反相 LC（RPLC）分离的糖肽进行保留时间归一化。串联质谱用于糖肽的检测和鉴定。此外，通过比较糖肽与其非糖基化对应物的保留时间，研究了不同类型聚糖对肽骨架疏水性的影响。基于肽保留时间校准混合物中标准肽的疏水性指数，评估了来自模型糖蛋白（包括牛胎球蛋白、α1-酸性糖蛋白和人血浆中的触珠蛋白）的胰蛋白酶消化糖肽的疏水性。由于亲水糖结构，多个肽骨架的疏水性降低。通过比较不同时间和仪器收集的糖肽的疏水性指数，该方法的日间和实验室间比较表明其具有高度的可靠性和可重复性。来自实验室间实验的疏水性指数的 RSD%为 1.2%，而保留时间的 RSD%为 5.1%。然后，该方法在从人血清中提取的复杂糖肽样品上的应用得到了验证。疏水性指数可用于解决使用不同仪器时的保留时间偏移问题，从而增强糖肽特征描述的信心。
