Symphogen, Pederstrupvej 93, 2750 Ballerup, Denmark.
Anal Chem. 2023 Oct 24;95(42):15465-15471. doi: 10.1021/acs.analchem.3c02609. Epub 2023 Oct 12.
Recently, we introduced an optimized and automated Multi-Attribute Method (MAM) workflow, which (a) significantly reduces the number of missed cleavages using an automated two-step digestion procedure and (b) dramatically reduces chromatographic peak tailing and carryover of hydrophobic peptides by implementing less retentive reversed-phase column chemistries. Here, further insights are provided on the impact of postdigest acidification and the importance of maintaining hydrophobic peptides in solution using strong chaotropic agents after digestion. We demonstrate how oxidation can significantly increase the solubility of hydrophobic peptides, a fact that can have a profound impact on quantitation of oxidation levels if care is not taken in MAM workflows. We conclude that (a) postdigestion acidification can result in significant acid-catalyzed deamidation during storage in an autosampler at 5 °C and (b) a strong chaotropic agent, such as guanidine hydrochloride, is critical for preventing loss of hydrophobic peptides through adsorption, which can result in (sometimes extreme) biases in quantitation of tryptophan oxidation levels. An optimized method is presented, which effectively addressed acid-catalyzed deamidation and solubility of hydrophobic peptides in MAM workflows.
最近，我们引入了一种经过优化和自动化的多属性方法（MAM）工作流程，该方法（a）通过自动化的两步消化程序显著减少了漏切的数量，（b）通过实施保留性较弱的反相柱化学物质，极大地减少了色谱峰拖尾和疏水性肽的残留。在这里，我们进一步深入探讨了消化后酸化的影响，以及在消化后使用强离液剂将疏水性肽保持在溶液中的重要性。我们证明了氧化如何显著提高疏水性肽的溶解度，如果在 MAM 工作流程中不加以注意，这一事实可能会对氧化水平的定量产生深远影响。我们得出结论：（a）消化后酸化会导致在 5°C 的自动进样器中储存时发生显著的酸催化脱酰胺，（b）强离液剂，如盐酸胍，对于防止疏水性肽通过吸附而损失至关重要，这可能导致（有时是极端的）色氨酸氧化水平定量的偏差。提出了一种优化的方法，该方法有效地解决了 MAM 工作流程中酸催化脱酰胺和疏水性肽溶解度的问题。
