Polar Aprotic Modifiers for Chromatographic Separation and Back-Exchange Reduction for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry |
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Authors: | Santosh?G?Valeja Mark?R?Emmett Email author" target="_blank">Alan?G?MarshallEmail author |
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Institution: | (1) Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, FL 32306, USA;(2) Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL 32310-4005, USA; |
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Abstract: | 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. |
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