Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203/8, 500 03 Hradec Králové, Czech Republic.
Anal Chem. 2024 Sep 10;96(36):14531-14540. doi: 10.1021/acs.analchem.4c02819. Epub 2024 Aug 28.
Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.
提高柱温是改善肽色谱分离的有效策略。然而，高温会诱导人为修饰，从而影响肽分析的质量。在这里，我们提出了一种新颖的高温 LC-MS 方法，该方法保留了高温柱温的优势，同时在反相液相色谱过程中显著减少了肽的修饰和降解。我们的方法利用了一个在线短捕集柱，该柱保持在接近环境温度，安装在分离柱的上游。捕集柱的保留性和尺寸经过优化，以缩短肽在加热分离柱中的停留时间，同时不影响分离性能。这种易于实施的方法在曲妥珠单抗的 110 分钟肽图中增加了 1.4 倍的峰容量，并在探索性 LC-MS 蛋白质组学分析中提供了 10%的肽鉴定，而与在 30°C 下进行的分析相比，同时保持修饰程度接近背景水平。在生物制药的肽图中，柱内修饰可能会错误地提高某些关键质量属性的水平，与直接在 60°C 下进样相比，该方法使 N 端焦谷氨酸和氧化 Met 的温度相关人为产物分别减少了 66%和 63%，从而提高了蛋白质药物质量控制的可靠性。我们的研究结果代表了 LC-MS 方法学的一个有前途的进展，为研究人员和行业专业人员提供了一种有价值的工具，用于改善肽的色谱分离，同时显著减少不必要的修饰。
