Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, United States.
J Proteome Res. 2024 Jan 5;23(1):409-417. doi: 10.1021/acs.jproteome.3c00627. Epub 2023 Nov 27.
A fast and sensitive direct extraction (DE) method developed in our group can efficiently extract proteins in 30 min from a 5 cm-long hair strand. Previously, we coupled DE to downstream analysis using gel electrophoresis followed by in-gel digestion, which can be time-consuming. In searching for a better alternative, we found that a combination of DE with a bead-based method (SP3) can lead to significant improvements in protein discovery in human hair. Since SP3 is designed for general applications, we optimized it to process hair proteins following DE and compared it to several other in-solution digestion methods. Of particular concern are genetically variant peptides (GVPs), which can be used for human identification in forensic analysis. Here, we demonstrated improved GVP discovery with the DE and SP3 workflow, which was 3 times faster than the previous in-gel digestion method and required significantly less instrument time depending on the number of gel slices processed. Additionally, it led to an increased number of identified proteins and GVPs. Among the tested in-solution digestion methods, DE combined with SP3 showed the highest sequence coverage, with higher abundances of the identified peptides. This provides a significantly enhanced means for identifying proteins and GVPs in human hair.
我们小组开发了一种快速灵敏的直接提取（DE）方法，能够在 30 分钟内从 5 厘米长的头发中有效提取蛋白质。之前，我们将 DE 与下游的凝胶电泳分析相结合，然后进行胶内消化，这可能很耗时。在寻找更好的替代方法时，我们发现 DE 与基于珠粒的方法（SP3）相结合可以显著提高头发中蛋白质的发现。由于 SP3 是为通用应用设计的，我们对其进行了优化，以在 DE 后处理头发蛋白质，并将其与其他几种溶液内消化方法进行了比较。特别关注的是遗传变异肽（GVPs），它们可用于法医分析中的人类识别。在这里，我们证明了 DE 和 SP3 工作流程可以改进 GVP 的发现，与之前的胶内消化方法相比，速度提高了 3 倍，根据处理的凝胶片数量，所需的仪器时间也大大减少。此外，它还增加了鉴定蛋白质和 GVP 的数量。在测试的溶液内消化方法中，DE 与 SP3 结合显示出最高的序列覆盖率，鉴定肽的丰度更高。这为鉴定头发中的蛋白质和 GVP 提供了一种显著增强的手段。
