Polar Aprotic Modifiers for Chromatographic Separation and Back-Exchange Reduction for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
| Autor: | Alan G. Marshall, Mark R. Emmett, Santosh G. Valeja |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Analytical chemistry
Mass spectrometry High-performance liquid chromatography Fourier transform ion cyclotron resonance Article Mass Spectrometry chemistry.chemical_compound Structural Biology Animals Humans Horses Spectroscopy Chromatography High Pressure Liquid Chromatography Reverse-Phase Chromatography Fourier Analysis Chemistry Deuterium Exchange Measurement Proteins Dimethylformamide Peptide Fragments Pyrrolidinones Solvent Supercritical fluid chromatography Hydrogen–deuterium exchange Hydrophobic and Hydrophilic Interactions |
| Popis: | Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein–protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|