Origin and number of charges observed on multiply-protonated native proteins produced by ESI

Abstract

Native proteins and particularly native non-covalently bonded protein–protein and protein–substrate complexes are of great interest and are intensely studied by ESI–MS methods. The multiple charges on these ions are not only useful in lowering the m/ z values but play also an important role in the chemical behavior of these complexes. Evidence from the literature and the present work is presented which supports the charge residue model (CRM) as the mode of formation of the charged globular proteins in the gas phase. Very small water droplets which contain only one protein molecule are ultimately formed in the ESI process. The surface of these droplets is charged by an excess of small ions due to a salt which is also present in the solution. Thus, in the positive ion mode, and when the buffer (ammonium acetate) is the main electrolyte used, the excess small positive ions are NH 4 + ions. Evaporation of the water in the droplet leads to a residue which is the globular protein. The protein is charged by the excess positive ions such as NH 4 +. The number of NH 4 + ions available, Z CRM, can be predicted on the basis of CRM. The proteins in order to be able to hold all of the protons provided must have a sufficient number of basic side chains located at the surface of the protein. It is found that most proteins have more than enough basic sites to hold the charge, Z CRM. Examples for these are carbonic anhydrase and cytochrome c. For these proteins the charge observed with ESI–MS is found to be close to equal to the charge, Z CRM, supplied. Some unusual proteins such as pepsin, have too few basic side chains, much less than the number of charges, Z CRM, provided. For these proteins the number of basic sites available on the protein determine how many of the charges provided by CRM will be retained. The number of basic sites can be evaluated and is found in agreement with the observed charges in the mass spectrum. Other predictions can also be made on the basis of the CRM. Thus, evaporation of the water droplet will lead to formation of neutral (uncharged) adducts on the protein, which are due to neutral components of the buffer. The approximate number of adducts can be predicted. Predictions can also be made which buffers will lead to adducts difficult to get rid off, in the desolvation stage of the mass spectrometer.