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

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

Author information

1
Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin, United States.
2
Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin, United States.
3
PolyLC, Inc., 9151 Rumsey Rd., Suite 180, Columbia, Maryland, United States.
4
Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, Wisconsin, United States.

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.

PMID:
26729044
PMCID:
PMC4947469
DOI:
10.1021/acs.analchem.5b04285
[Indexed for MEDLINE]
Free PMC Article

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