Polar aprotic modifiers for chromatographic separation and back-exchange reduction for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.
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.
Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, FL 32306, USA.
Santosh G
SG
Valeja
Mark R
MR
Emmett
Alan G
AG
Marshall
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004126:Dimethylformamide / Q000737:chemistry; D005583:Fourier Analysis; D006736:Horses; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011760:Pyrrolidinones / Q000737:chemistry
D010446:Peptide Fragments; D011506:Proteins; D011760:Pyrrolidinones; D004126:Dimethylformamide; C038678:N-methylpyrrolidone