American Chemical Society
ac1016858_si_001.pdf (141.41 kB)
Download file

New Reagents for Enhanced Liquid Chromatographic Separation and Charging of Intact Protein Ions for Electrospray Ionization Mass Spectrometry

Download (141.41 kB)
journal contribution
posted on 2010-09-01, 00:00 authored by Santosh G. Valeja, Jeremiah D. Tipton, Mark R. Emmett, Alan G. Marshall
Electrospray ionization produces multiply charged ions, thereby lowering the mass-to-charge ratio for peptides and small proteins to a range readily accessed by quadrupole ion trap, orbitrap, and ion cyclotron resonance (ICR) mass analyzers (m/z = 400−2000). For Fourier transform mass analyzers (orbitrap and ICR), higher charge also improves signal-to-noise ratio, mass resolution, and mass accuracy. Addition of m-nitrobenzyl alcohol (m-NBA) or sulfolane has previously been shown to increase the charge states of proteins. Moreover, polar aprotic dimethylformamide (DMF) improves chromatographic separation of proteolytic peptides for mass analysis of solution-phase protein hydrogen/deuterium exchange for improved (78−96%) sequence coverage. Here, we show that addition of each of the various modifiers (DMF, thiodiglycol, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone) can significantly increase the charge states of proteins up to 78 kDa. Moreover, incorporation of the same modifiers into reversed-phase liquid chromatography solvents improves sensitivity, charging, and chromatographic resolution for intact proteins.