Charge state assignment from schiff‐base adducts in low resolution electrospray mass spectra of protein mixtures and dissociation products

Schiff‐bases formed by reaction between ketones and free amine sites in proteins yield mass‐shifted ions that are useful in assigning charge states in low resolution electrospray ionization mass spectra of mixtures and dissociation products. The technique is attractive for those mass analyzers incapable of achieving sufficient resolving power to distinguish the 13C‐isotope distribution within an individual charge state. Instead, acetone is used here to append a C3H4 group to the protein and achieve a forty‐fold reduction in the necessary mass resolution for charge state determination. The utility of Schiff‐base adducts for assigning charge states among a mixture of proteins is demonstrated; here, the molecular weights for horse myoglobin (MW 16 950) and cytochrome c (MW 12 360) in a two component mixture are unambiguously determined from a mass spectrum with a mass resolution of only 800. The CN bond formed by the Schiff base is sufficiently strong to survive ion dissociation processes and permit assignment of charge state to fragment ions in tandem mass spectrometry experiments. For example, adducted sequence ions are observed in high yield when a melittin/acetone solution is subjected to nozzle‐skimmer dissociation conditions in the electrospray process. Other advantages of Schiff‐base reaction include site‐specificity and minimal alteration to the charge state. This site‐specific binding feature appears to be promising method for locating the lysine groups in structural characterization by tandem mass spectrometry. Copyright © 1995 John Wiley & Sons, Ltd.

Duke Scholars

Author Ziqiang Guan Biochemistry