Ion-pair Reversed-phase×Low-pH Reversed-phase Two-dimensional Liquid Chromatography for In-depth Proteomic Profiling

doi:10.1007/s40242-022-2166-y

Abstract

Abstract: High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples. Herein, we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid chromatography (IPRP×RP 2DLC) strategy for comprehensive proteomic analysis. Both RPLC separation dimensions were performed at low pH, with trifluoroacetic acid(TFA) and formic acid(FA) as mobile phase addictive, respectively. As the good separation resolution offered by ion-pairing effect of TFA, the fractionation efficiency was greatly improved with 74.0% peptides identified in just one fraction. Comparing with conventional high pH RP fractionation, the overall separation rate of IPRP was about 1.6 times that of high-pH RP, which increased the number of identified peptides by 21%. Further, 2169 proteins and 8540 peptides were confidently identified from crude serum sample by our IPRP×RP 2DLC strategy, exhibiting great potential in clinical proteomics in the future.

Key words: Two-dimensional liquid chromatography(2DLC), Ion-pair reversed-phase liquid chromatography(IPRP-LC), IPRP×low-pH RP 2DLC, Proteomics, Mass spectrometry

NIU Wenxue, LIU Zheyi, LIU Jing, LAI Can, ZHANG Tingting, ZHAO Heng, WANG Guosheng, WANG Fangjun. Ion-pair Reversed-phase×Low-pH Reversed-phase Two-dimensional Liquid Chromatography for In-depth Proteomic Profiling[J]. Chemical Research in Chinese Universities, 2023, 39(2): 260-265.

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