School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30, Quai Ernest-Ansermet, 1211 Geneva 4, Switzerland.
J Chromatogr A. 2012 Oct 12;1259:187-99. doi: 10.1016/j.chroma.2012.05.033. Epub 2012 May 14.
The high resolution profiling of complex mixtures is indispensable for obtaining online structural information on the highest possible number of the analytes present. This is particularly relevant for natural extracts, as for the venom of the predatory marine snail Conus consors, which contains numerous bioactive peptides with molecular masses ranging between 1000 and 5000 Da. The goal of the present work was to maximise peak capacity of peptides separations by LC-MS while maintaining a reasonable analysis time. The best gradient performance using the C. consors venom as a real sample was obtained with a mobile phase flow rate as high as possible to maximise performance in the gradient mode, and gradient time comprised between 75 and 350 min when using a 150 mm column length. The present study also confirmed that an elevated temperature (up to 90 °C) improves performance under ultra-high pressure liquid chromatography (UHPLC) conditions. However, the thermal stability of the analytes had to be critically evaluated. For the profiling of C. consors, analyte degradation was not clearly observable at 90 °C with analysis times of approximately 100 min. Finally, the MS source was found to cause significant additional band broadening in the UHPLC mode (σ(ext)(2) was 10-24 times higher using TOF-MS vs. UV detection). Thus, if the MS contributes strongly to the peak capacity loss, classical 2.1mm I.D. columns can be replaced by 3.0mm I.D. to mitigate this problem. Based on these considerations, the optimal generic profiling conditions applied to the C. consors venom provided a peak capacity higher than 1100 for a gradient time of around 100 min, doubling the values reached by classical HPLC separation. UHPLC-QTOF-MS/MS experiments carried out in these conditions provided exploitable data that matched with peptides present in the C. consors venom. These optimal LC conditions are thus compatible with online peptide deconvolution and matching against transcriptomic data and, to some extent, de novo sequencing in such complex mixtures.
复杂混合物的高分辨率剖析对于获取存在的分析物的最高数量的在线结构信息是必不可少的。对于天然提取物来说，这一点尤其重要，因为捕食性海洋蜗牛 Conus consors 的毒液中含有许多具有 1000 至 5000 Da 分子量的生物活性肽。本工作的目的是通过 LC-MS 最大限度地提高肽分离的峰容量，同时保持合理的分析时间。使用 C. consors 毒液作为真实样品时，使用尽可能高的流动相流速可获得最佳的梯度性能，当使用 150mm 柱长时，梯度时间在 75 至 350min 之间。本研究还证实，在超高效液相色谱 (UHPLC) 条件下，升高温度（高达 90°C）可提高性能。然而，必须仔细评估分析物的热稳定性。对于 C. consors 的剖析，在 90°C 下分析时间约为 100min 时，没有明显观察到分析物降解。最后，发现 MS 源在 UHPLC 模式下会导致明显的附加带宽变宽（与 UV 检测相比，TOF-MS 的σ(ext)(2)高 10-24 倍）。因此，如果 MS 对峰容量损失有很大影响，则可以用 3.0mmID 代替经典的 2.1mmID 柱来解决此问题。基于这些考虑，应用于 C. consors 毒液的最佳通用剖析条件提供了超过 1100 的峰容量，对于大约 100min 的梯度时间，是经典 HPLC 分离值的两倍。在这些条件下进行的 UHPLC-QTOF-MS/MS 实验提供了可利用的数据，这些数据与 C. consors 毒液中的肽匹配。因此，这些最佳 LC 条件与在线肽拆卷积和与转录组数据匹配以及在这种复杂混合物中进行某种程度的从头测序兼容。
