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

Anal Chem. 2016 Feb 2;88(3):1885-91. doi: 10.1021/acs.analchem.5b04285. Epub 2016 Jan 14.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Aprotinin / analysis
  • Cattle
  • Chickens
  • Chromatography
  • Chymotrypsinogen / analysis
  • Horses
  • Hydrophobic and Hydrophilic Interactions*
  • Internet*
  • Lactoglobulins / analysis
  • Mass Spectrometry*
  • Muramidase / analysis
  • Muramidase / metabolism
  • Proteomics / methods*
  • Ribonuclease, Pancreatic / analysis
  • Ribonuclease, Pancreatic / metabolism
  • Trypsinogen / analysis

Substances

  • Lactoglobulins
  • Trypsinogen
  • Chymotrypsinogen
  • Aprotinin
  • Ribonuclease, Pancreatic
  • Muramidase