Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra

Abstract

Electrospray ionization produces multiply charged molecular ions for biomolecules with molecular weights in excess of 100,000. This allows mass spectrometers with limited mass-to-charge range to extend their molecular weight range by a factor equal to the number of charges. The maximum number of observed charges for peptides and smaller proteins correlates well with the number of basic amino acid residues (Arg, Lys, His), except for disulfide-containing molecules, such as lysozyme and bovine albumin. However, reduction of disulfide linkages with 1,4-dithiothreitol (Cleland's reagent) may allow the protein to be in an extended conformation and make "buried" basic residues available for protonation to yield higher charged molecular ions by the electrospray ionization process. For larger proteins reduction of disulfide bridges greatly increases the maximum charge state, but charging of basic amino acid residues remains less efficient than for smaller proteins.