Analysis of protein mixtures by electrospray mass spectrometry: effects of conformation and desolvation behavior on the signal intensities of hemoglobin subunits.
The determination of solution-phase protein concentration ratios based on ESI-MS intensity ratios is not always straightforward. For example, equimolar mixtures of hemoglobin alpha- and beta-subunits consistently result in much higher peak intensities for the alpha-chain. The current work explores the origin of this effect. Under mildly acidic conditions (pH 3.4) alpha-globin is extensively unfolded, whereas beta-globin retains residual structure. Because of its greater nonpolar character, the more unfolded alpha-subunit can more effectively compete for charge. This leads to suppression of beta-globin signals under conditions where the protein ion yield is limited by the charge concentration on the initially formed ESI droplets. More balanced intensities are observed when operating under charge excess conditions and/or in a solvent environment where both proteins are unfolded to a similar degree (pH 2.2). However, even in these cases the overall alpha-globin peak intensity is still twice as high as that of the beta-subunit. The persistent imbalance under these conditions originates from the different declustering behaviors of the two proteins. A considerable fraction of beta-globin undergoes incomplete desolvation during ESI, thereby reducing the intensity of bare [beta + zH](z+) ions. When including the contributions of incompletely desolvated species, the overall alpha:beta ion intensity ratio is close to unity. The alpha:beta intensity imbalance can also be eliminated by a strongly elevated declustering potential in the ion sampling interface. In conclusion, important factors that have to be considered for the ESI-MS analysis of protein mixtures are (1) conformational effects, resulting in differential surface activities, and (2) dissimilarities in the protein desolvation behavior.
Kuprowski
Mark C
MC
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
Boys
Brian L
BL
Konermann
Lars
L
D045424:Complex Mixtures / Q000032:analysis / Q000737:chemistry*; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D021122:Protein Subunits; D012997:Solvents / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D045424:Complex Mixtures; D006454:Hemoglobins; D021122:Protein Subunits; D012997:Solvents