Analytical Chemistry group, van't Hoff Insititute for Molecular Sciences (HIMS), University of Amsterdam, Science Park 904, 1098XH, Amsterdam, the Netherlands; Centre for Analytical Sciences Amsterdam, van't Hoff Insititute for Molecular Sciences (HIMS), University of Amsterdam, Science Park 904, 1098XH, Amsterdam, the Netherlands.
Analytical Chemistry group, van't Hoff Insititute for Molecular Sciences (HIMS), University of Amsterdam, Science Park 904, 1098XH, Amsterdam, the Netherlands; Centre for Analytical Sciences Amsterdam, van't Hoff Insititute for Molecular Sciences (HIMS), University of Amsterdam, Science Park 904, 1098XH, Amsterdam, the Netherlands.
Anal Chim Acta. 2023 Jul 25;1266:341324. doi: 10.1016/j.aca.2023.341324. Epub 2023 May 3.
Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-μL/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-salt concentrations up to 400 mM. Band broadening and loss of resolution resulting from the introduction of injection volumes exceeding 3% of the column volume could be circumvented by incorporating an online trap-column containing a mixed-bed ion-exchange (IEX) material. The online IEX-based solid-phase extraction (SPE) or "trap-and-elute" set-up provided on-column focusing (sample preconcentration). This allowed the injection of large sample volumes on the 1-mm I.D. SEC column without compromising the separation. The enhanced sensitivity attained by the micro-flow SEC-MS, along with the on-column focusing achieved by the IEX precolumn, provided picogram detection limits for proteins.
体积排阻色谱（SEC）采用中性 pH 值下含有挥发性盐的水性流动相，并结合天然质谱（nMS），是一种用于表征天然状态下蛋白质和蛋白质聚集体的有价值的工具。然而，SEC-nMS 中经常使用的液相条件（高盐浓度）阻碍了对气相中不稳定蛋白质复合物的分析，需要更高的去溶剂化气流和源温度，导致蛋白质碎片化/解离。为了解决这个问题，我们研究了在 15 μL/min 流速下运行的窄 SEC 柱（内径为 1.0 毫米）及其与 nMS 的耦合，用于蛋白质、蛋白质复合物和高级结构（HOS）的表征。降低的流速显著增加了蛋白质离子化效率，便于检测低丰度杂质和高达 230 kDa 的 HOS（即使用的 Orbitrap-MS 仪器的上限）。更有效的溶剂蒸发和更低的去溶剂化能量允许更软的离子化条件（例如，更低的气体温度），从而确保在转移到气相过程中，蛋白质及其 HOS 几乎没有或没有结构改变。此外，洗脱盐的离子抑制作用降低，允许使用高达 400 mM 的挥发性盐浓度。通过引入超过柱体积 3%的进样体积，可能会导致峰展宽和分辨率降低，这可以通过包含含有混合床离子交换（IEX）材料的在线捕集柱来避免。在线 IEX 基于固相萃取（SPE）或“捕集和洗脱”设置提供了柱上聚焦（样品预浓缩）。这允许在 1 毫米内径 SEC 柱上注入大体积的样品，而不会影响分离。微流速 SEC-MS 获得的增强灵敏度以及 IEX 预柱实现的柱上聚焦，为蛋白质提供了皮克级检测限。
