Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
Clinical Proteomics Core Laboratory, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan.
J Chromatogr A. 2018 Oct 12;1571:201-212. doi: 10.1016/j.chroma.2018.08.020. Epub 2018 Aug 10.
Reverse-phase (RP) liquid chromatography (RPLC) and size-exclusion chromatography (SEC) are methods commonly used for protein/peptide separation, and they are based on distinct principles. This study develops a method using RP columns for size-based separation of protein mixtures. Results show that high concentrations of acetonitrile with trifluoroacetic acid as an acid modifier successfully suppressed interactions between proteins and the stationary phase and allowed the RP column to act as a SEC column to separate proteins based on their molecular weight. The reduction of protein disulfide bonds resulted in an improved correlation between the retention time and molecular weight in the RP-based SEC, which indicates that conformation-dependent SEC retention is less important for disulfide-reduced proteins using a current mobile phase. Importantly, the employed salt-free mobile phase allowed the RP-based SEC system to be directly coupled with online mass spectrometry (MS) analysis. Furthermore, by reducing the flow rate and increasing the column length, the separation efficiency was further improved and no adverse effects due to the prolonged separation were observed, which indicates the potential of this strategy to serve as a first-dimensional separation method for constructing an online multi-dimensional LC system. The size-based separation phenomenon with RP columns was further evaluated using a complex protein mixture (a cell lysate), and compared to conventional SEC, more proteins of the cell lysate were observed following the SEC separation principle, which implies the better generality of usage of the RP-based SEC method for protein separation. In summary, results show that the RP-based SEC is highly efficient in achieving protein fractionation. In addition, the employed salt-free mobile phase provides excellent compatibility of the RP-based SEC with other separation strategies or online mass spectrometric analysis. We anticipate that laboratories using RP-HPLC for protein separation will easily be able to move to the size-based separation mode using the same RP-HPLC system.
反相（RP）液相色谱（RPLC）和尺寸排阻色谱（SEC）是常用于蛋白质/肽分离的方法，它们基于不同的原理。本研究开发了一种使用 RP 柱基于大小分离蛋白质混合物的方法。结果表明，高浓度的乙腈与三氟乙酸作为酸改性剂成功地抑制了蛋白质与固定相之间的相互作用，使 RP 柱能够作为 SEC 柱根据分子量分离蛋白质。蛋白质中二硫键的减少导致在基于 RP 的 SEC 中保留时间与分子量之间的相关性得到改善，这表明在当前流动相下，构象依赖性 SEC 保留对于还原二硫键的蛋白质不太重要。重要的是，所采用的无盐流动相允许基于 RP 的 SEC 系统直接与在线质谱（MS）分析相耦合。此外，通过降低流速和增加柱长，进一步提高了分离效率，并且没有观察到由于分离时间延长而产生的不利影响，这表明该策略有潜力作为在线多维 LC 系统构建的一维分离方法。使用复杂的蛋白质混合物（细胞裂解物）进一步评估了基于 RP 的 SEC 的基于大小的分离现象，并与常规 SEC 相比，在 SEC 分离原理之后观察到更多的细胞裂解物中的蛋白质，这意味着基于 RP 的 SEC 方法在蛋白质分离方面的使用具有更好的通用性。总之，结果表明基于 RP 的 SEC 在实现蛋白质分级方面非常高效。此外，所采用的无盐流动相提供了基于 RP 的 SEC 与其他分离策略或在线质谱分析的出色兼容性。我们预计，使用 RP-HPLC 进行蛋白质分离的实验室将能够轻松地将其转换为使用相同的 RP-HPLC 系统的基于大小的分离模式。
