Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics

详细信息    查看全文

• 作者：Bifan Chen ; Ying Peng ; Santosh G. Valeja ; Lichen Xiu ; Andrew J. Alpert ; Ying Ge
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：February 2, 2016
• 年：2016
• 卷：88
• 期：3
• 页码：1885-1891
• 全文大小：466K
• ISSN：1520-6882

文摘

Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics.