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

نویسندگان

  • Bifan Chen
  • Ying Peng
  • Santosh G Valeja
  • Lichen Xiu
  • Andrew J Alpert
  • Ying Ge
چکیده

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.

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عنوان ژورنال:
  • Analytical chemistry

دوره 88 3  شماره 

صفحات  -

تاریخ انتشار 2016