Predicting the highest intensity ion in multiple charging envelopes observed for denatured proteins during electrospray ionization mass spectrometry by inspection of the amino acid sequence.

A simple, manual method for predicting the highest intensity charge states (HICS) of denatured protein ions generated by electrospray ionization based on inspection of the proteins' amino acid sequence is proposed. The HICS is accurately predicted by identifying groupings of nearby basic amino acids in the positive mode or acidic amino acid residues in the negative mode. The method assumes that the likelihood of having more than one charge per group becomes less likely due to Coulombic repulsion of like charges. It is shown empirically that a spacing of at least three noncharged residues is required between charged amino acids for the charge state with the highest intensity. Verification of this method is presented, and its limitations are identified. It is fast, inexpensive, and provides similar, although less detailed, information as state-of-the-art methods that rely on computational calculations. With a few exceptions, the highest intensity charge states were predicted to an average of one charge state of the experimental data. For those proteins whose HICS were not accurately estimated, the experimental values fell short of the predictions. Upon reduction of the disulfide bonds of these proteins, the experimental HICS became closer to the predicted values, suggesting that charging lower than the prediction can be attributed to conformational inflexibility of those proteins.