On the role of a direct interaction between protein ions and solvent additives during protein supercharging by electrospray ionization mass spectrometry.

The addition of certain reagents during the electrospray ionization mass spectrometry of proteins can shift the protein ion signal charge-state distributions (CSDs) to higher average charge states, a phenomenon known as 'supercharging'. The role of reagent gas-phase basicity (GB) during this process was investigated in both the negative and positive ion modes. Reagents with known or calculated GBs were added individually in equimolar amounts to protein solutions which were subsequently electrosprayed for mass spectrometry analysis. Shifts in the CSDs of the protein ion signals were monitored and related to the reagents' GBs. Trends for this data were evaluated for possible insights into a supercharging mechanism involving the direct interaction between supercharging reagent and protein ion. Reagent GB was confirmed to be directly related to the amount of supercharging observed in the negative ion mode. Supercharging in the positive ion mode, on the other hand, showed a maximal trend. Interestingly, a loss of signal and supercharging efficacy was observed for reagents with GBs intermediate within the investigated range, between ~800 and ~840 kJ mol(-1), at the 100 mM concentration used in the present study. The possibility of a direct interaction model for supercharging in the negative and positive ion modes dependent on the GBs of the protein ions and reagents is discussed. In the positive ion mode, supercharging appears to depend on the stability of a proton bridge formed between the reagent and a highly charged protein ion.

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.

Deconstructing desorption electrospray ionization: independent optimization of desorption and ionization by spray desorption collection.

Spray desorption collection (SDC) and reflective electrospray ionization (RESI) were used to independently study the desorption and ionization processes that together comprise desorption electrospray ionization (DESI). Both processes depend on several instrumental parameters, including the nebulizing gas flow rate, applied potential, and source geometries. Each of these parameters was optimized for desorption, as represented by the results obtained by SDC, and ionization, as represented by the results obtained by RESI. The optimized conditions were then compared to the optimization results for DESI. Our results confirm that optimal conditions for desorption and ionization are different and that in some cases the optimized DESI conditions are a compromise between both sets. The respective results for DESI, RESI, and SDC for each parameter were compared across the methods to draw conclusions about the contribution of each parameter to desorption and ionization separately and then combined within DESI. Our results indicate that desorption efficiency is (1) independent of the applied potential and (2) the impact zone to inlet distance, and that (3) gas pressure settings and (4) sprayer to impact zone distances above optimal for DESI are detrimental to desorption but beneficial for ionization. In addition, possible interpretations for the observed trends are presented.