Ultra-High Pressure Fast Size Exclusion Chromatography for Top-Down Proteomics

摘要

Top-down mass spectrometry (MS)-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the liquid chromatographic separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. Size exclusion chromatography (SEC) is a favored liquid chromatography method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein we reported the use of ultra-high pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6 – 669 kDa) was achieved in less than 7 min with high-resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size-separation of proteins with great potential for top-down proteomics.