Bioprocess Measurements Group, Biomolecular Measurement Division, National Institute of Standards and Technology, Rockville, Maryland 20850, United States.
Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, Maryland 20850, United States.
J Am Soc Mass Spectrom. 2023 Dec 6;34(12):2672-2679. doi: 10.1021/jasms.3c00243. Epub 2023 Nov 6.
Chromatographic separations at subzero temperature significantly improve the precision of back-exchange-corrected hydrogen-deuterium exchange mass spectrometry (HDX-MS) determinations. Our previously reported dual-enzyme HDX-MS analysis instrument used reversed phase liquid chromatography (RPLC) at -30 °C, but high backpressures limited flow rates and required materials and equipment rated for very high pressures. Here, we report the design and performance of a dual-enzyme HDX-MS analysis instrument comprising a RPLC trap column and a hydrophilic interaction liquid chromatography (HILIC) analytical column in a two-dimensional RPLC-HILIC configuration at subzero temperature. During operation at -30 °C, the HILIC column manifests greatly reduced backpressure, which enables faster analytical flow rates and the use of materials rated for lower maximum pressures. The average peptide eluted from a HILIC column during a 40 min gradient at -30 °C contained ≈13% more deuterium than peptides eluted from a tandem RPLC-RPLC apparatus using a conventional 8 min gradient at 0 °C. A subset of peptides eluted from the HILIC apparatus contained ≈24% more deuterium.
在零下温度下进行色谱分离可显著提高反向交换校正氢氘交换质谱（HDX-MS）测定的精度。我们之前报道的双酶 HDX-MS 分析仪器在-30°C 下使用反相液相色谱（RPLC），但高压会限制流速，需要使用能够承受超高压力的材料和设备。在这里，我们报告了一种双酶 HDX-MS 分析仪器的设计和性能，该仪器由 RPLC 捕获柱和在零下温度下的二维 RPLC-HILIC 配置中的亲水相互作用液相色谱（HILIC）分析柱组成。在-30°C 下运行时，HILIC 柱的背压大大降低，这使得分析流速更快，并可使用承受较低最大压力的材料。在-30°C 下进行 40 分钟梯度洗脱时，从 HILIC 柱洗脱的平均肽的氘含量比在 0°C 下使用传统的 8 分钟梯度洗脱的串联 RPLC-RPLC 装置洗脱的肽多约 13%。从 HILIC 装置洗脱的一部分肽的氘含量多约 24%。
