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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.

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