Chromatographic efficiency and selectivity in top-down proteomics of histones

Abstract

Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-minute gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5 μm in diameter, 2) superficially porous particles of 3 μm in diameter with a shell of 0.2 μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics

“…Much effort has been made to improve the separation of proteoforms with RPLC. Monolithic columns and packed columns with beads having various sizes, different lengths of carbon chains and varied porosity have been investigated for proteoform separation [4,[7][8][9]. However, several challenges remain for top-down proteomics, including high-capacity separations of proteoforms and achieving informative MS/MS dissociation of proteoforms.…”

A perspective view of top‐down proteomics in snake venom research

“…Therefore, other well‐known separation methods based on hydrophobicity (hydrophobic interaction chromatography [HIC] and hydrophilic interaction [HILIC]), charge (ion‐exchange chromatography [IEX] and mixed‐mode chromatography [MMC]) and size (size‐exclusion chromatography [SEC]) have been used for a better isolation and resolution of proteoforms prior to MS. In spite of the fact that LC approaches remain the desired methods for online intact protein separation, several electrophoretic techniques including isoelectric focusing (IEF), gel elution liquid fraction entrapment electrophoresis (GELFrEE), capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) have been described as potential alternatives. The application of different separation techniques yields a variety of results, but none of them are solely able to provide a high‐resolution separation especially for complex proteomes.…”

Proteins and Proteoforms: New Separation Challenges

“…When used in histone proteoform separations with TFA, a C18 slip flow column was superior to RPC columns packed with fully porous 5 µm particles or superficially porous 3 µm particles. 146 It was also noted that TFA caused less band spreading than difluoroacetic acid. Proteoforms of ribonuclease A, carbonic anhydrase, superoxide dismutase, and trypsin inhibitor were resolved with the slip flow RPC column.…”

“…The peak capacity of this system increased to 750 in a 60 min gradient. When used in histone proteoform separations with TFA, a C18 slip flow column was superior to RPC columns packed with fully porous 5 μm particles or superficially porous 3 μm particles . It was also noted that TFA caused less band spreading than difluoroacetic acid.…”