Column heating strategy is often applied in nano-high-performance liquid chromatography-mass spectrometer (nanoHPLC-MS) platform for enhancing the analytical efficiency of peptides or proteins. Nonetheless, the influence effects of column heating in peptides or proteins identification still lack of deep understanding. In this study, a systematic comparison of room temperature (RT) and column heating of nanoHPLC was done. Based on the data, under column heating condition, the backpressure of nanoHPLC can be decreased. Due to the increase of resolution, the peak widths of precursor ion were narrowed. As a result, in MS/MS data acquisition part, more time was spared for MS1 detecting and MS2 fragmenting, which eventually resulted in increased identification of peptides and proteins. Moreover, we also proposed the application scope of column heating by evaluating its influence on sample detection. On one hand, column heating significantly increased the identification of membrane proteins due to more efficient elution of highly hydrophobic peptides compared with RT. On the other hand, heating was not suitable for analyzing short or/and hydrophilic peptides with low retention time, which would be eluted out during sample loading process under high temperature and missed by mass spectrometric detection. In conclusion, our study provides a reference for rational application of column heating in proteomics research.

中文翻译：

---

基于纳流液相色谱质谱（nanoLC-MS）的蛋白质组学中柱加热效果的表征。

色谱柱加热策略通常用于纳米高性能液相色谱-质谱仪（nanoHPLC-MS）平台中，以提高肽或蛋白质的分析效率。尽管如此，柱加热对肽或蛋白质鉴定的影响仍然缺乏深刻的理解。在这项研究中，对室温（RT）和nanoHPLC的柱加热进行了系统的比较。根据这些数据，在柱加热条件下，可以降低nanoHPLC的背压。由于分离度的提高，前驱体离子的峰宽变窄。结果，在MS / MS数据采集部分，为MS1检测和MS2片段化腾出了更多时间，最终导致了对肽和蛋白质的鉴定增加。而且，我们还通过评估柱加热对样品检测的影响，提出了柱加热的应用范围。一方面，由于与RT相比，高疏水性肽的洗脱效率更高，因此柱加热显着提高了膜蛋白的鉴定。另一方面，加热不适用于分析具有短保留时间的短肽和/或亲水肽，在高温下的上样过程中，洗脱的肽会被洗脱，而质谱检测会丢失。总之，我们的研究为蛋白质组学研究中柱加热的合理应用提供了参考。另一方面，加热不适用于分析具有短保留时间的短肽和/或亲水肽，在高温下的上样过程中，洗脱的肽会被洗脱，而质谱检测会丢失。总之，我们的研究为蛋白质组学研究中柱加热的合理应用提供了参考。另一方面，加热不适合分析保留时间短的短肽和/或亲水肽，在高温下的样品上样过程中洗脱出的肽会被质谱检测遗漏。总之，我们的研究为蛋白质组学研究中柱加热的合理应用提供了参考。