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

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 non-denaturing 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 mass spectrometry. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics.

Top-down Proteomics: Technology Advancements and Applications to Heart Diseases

“…In HIC, a gradient with a decreasing concentration of salt is used to separate proteins based on hydrophobic interactions. Commonly used salts for achieving high-resolution separation of proteins by HIC contains sulfate, phosphate, and citrate, which are incompatible with MS [45]. Ammonium acetate is an MS-compatible salt, but HIC separation using ammonium acetate suffers from low separation power due to inadequate protein retention when used with conventional HIC materials [45].…”

“…recently evaluated a series of HIC columns with heightened degrees of hydrophobicity to compensate for the inadequate retention of proteins by the stationary phase when using ammonium acetate in the mobile phase. They demonstrated improved protein retention using columns with stationary phases that were more hydrophobic than conventional HIC stationary phases and a mobile phase containing ammonium acetate, which opens the door for the use of this powerful high-resolution separation method for top-down proteomics analysis [45]. Importantly, HIC and RPC are orthogonal with different selectivity for intact protein separation [46], despite the fact that both methods separate proteins based on hydrophobicity.…”

Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations

“…Most commonly used are moderately hydrophobic ligands such as n -alkanes (butyl, octyl, phenyl) [82, 88]. However, newly developed materials have emerged which use cholesterol [89], dendronic ligands [90] and dual functional stationary phases [91].…”

Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes

“…Online HIC− MS as well as HIC−RPC−MS have been successfully employed for top-down proteomics analyses of single proteins and complex protein mixtures. 19,20 We considered HIC to be a promising alternative to already established fractionation techniques for studying single proteins and protein complexes by bottom-up MS-based proteomics. The developed workflow is suitable for characterization of native soluble protein complexes as well as peptide mapping of single purified proteins and biologics.…”