Oyler Jonathan, Sullivan Raymond F, Tran Bao Q, Baker Dhanwin, Coveney Clare, Boocock David, Oyler Benjamin, Perry Carole C, Kilgour David P A
Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom.
U.S. Army DEVCOM-CCDC Chemical Biological Center, 5100 Blackhawk Rd., Building E3330, Aberdeen Proving Ground, Gunpowder, MD 21010, USA.
J Proteomics. 2025 Feb 20;312:105359. doi: 10.1016/j.jprot.2024.105359. Epub 2024 Nov 28.
Limitations to many current aqueous-based tryptic digestion methods include lengthy digestion times and both relatively high inter- and intra-day variability for both characteristic peptides identified and sequence coverages. This report describes results from digestion of some complex biomedical samples using the rapid Denaturing Organic Digestion method (DOD), an organic solvent-modified digestion method previously optimized for targeted protein digestion. Advantages of the DOD method included a very rapid digestion only requiring inexpensive solvents and reagents generally available in the laboratory, with no requirement for specialized equipment or expensive, specialized consumables. For this study, samples of E. coli and murine ileum protein extracts, and K562, a mass spectrometry-compatible human protein extract and reference standard routinely used to evaluate methods, were digested. Sequence coverage and characteristic peptide identification results were compared to those from 18 and 24 h conventional aqueous-based digestion methods. Across the samples tested, though the number of characteristic peptides and sequence coverages produced by the 5 min DOD method were very similar to those produced by the aqueous-based digestion methods, the specific characteristic proteins and their corresponding tryptic peptides identified following DOD method digestion included more hydrophilic and less hydrophobic species. In addition, we explored the effect of increasing digestion times with complex samples from 5 to 30 and 90 min for the DOD method. Increasing the digestion time to ≥30 min resulted in improved intra-day precision and the identification of many more peptide products than the currently used aqueous methods to which it was compared. These results suggest that the DOD organic-modified digestion method could, while markedly reducing protein digestion time, also provide more precise analysis and access to a somewhat different area of the proteome than that provided by current aqueous-based digestion methods. SIGNIFICANCE: The DOD tryptic digest method is a very simple and rapid process with no requirement for expensive equipment or consumables. The method markedly reduces tryptic digestion time and cost, and substantially improves within-batch and across-analyst precision for peptide and sequence coverage results over methods to which it was compared. Importantly, it also provides access to a somewhat different subset of the proteome with different peptide products identified as compared to aqueous solvent-based digestion providing potential for increased proteome coverage for bottom-up analysis if used in conjunction with aqueous-based methods.
许多当前基于水相的胰蛋白酶消化方法存在局限性，包括消化时间长，以及所鉴定的特征肽和序列覆盖率在日间和日内的变异性都相对较高。本报告描述了使用快速变性有机消化法（DOD）对一些复杂生物医学样品进行消化的结果，DOD是一种先前针对靶向蛋白质消化进行优化的有机溶剂改良消化方法。DOD方法的优点包括消化速度非常快，只需要实验室中通常可用的廉价溶剂和试剂，无需专门设备或昂贵的专用耗材。在本研究中，对大肠杆菌和小鼠回肠蛋白提取物样品，以及K562（一种与质谱兼容的人蛋白提取物和常用于评估方法的参考标准品）进行了消化。将序列覆盖率和特征肽鉴定结果与18小时和24小时的传统水相消化方法的结果进行了比较。在所测试的样品中，尽管5分钟DOD方法产生的特征肽数量和序列覆盖率与水相消化方法产生的非常相似，但DOD方法消化后鉴定出的特定特征蛋白及其相应的胰蛋白酶肽包括更多亲水性和更少疏水性的物种。此外，我们探讨了将复杂样品的DOD方法消化时间从5分钟增加到30分钟和90分钟的效果。将消化时间增加到≥30分钟可提高日内精密度，并且与目前使用的水相方法相比，可鉴定出更多的肽产物。这些结果表明，DOD有机改良消化方法在显著缩短蛋白质消化时间的同时，还能提供比当前水相消化方法更精确的分析，并能获取蛋白质组中略有不同的区域。意义：DOD胰蛋白酶消化方法是一个非常简单快速的过程，无需昂贵的设备或耗材。该方法显著缩短了胰蛋白酶消化时间和成本，与所比较的方法相比，大幅提高了肽和序列覆盖率结果的批内和不同分析人员间的精密度。重要的是，与基于水相溶剂的消化相比，它还能获取蛋白质组中略有不同的子集，鉴定出不同的肽产物，如果与水相方法结合使用，有可能增加自下而上分析的蛋白质组覆盖率。
