Direct correlation of the crystal structure of proteins with the maximum positive and negative charge states of gaseous protein ions produced by electrospray ionization

Abstract

Electrospray mass spectrometric studies in native folded forms of several proteins in aqueous solution have been performed in the positive and negative ion modes. The mass spectra of the proteins show peaks corresponding to multiple charge states of the gaseous protein ions. The results have been analyzed using the known crystal structures of these proteins. Crystal structure analysis shows that among the surface exposed residues some are involved in hydrogen-bonding or salt-bridge interactions while some are free. The maximum positive charge state of the gaseous protein ions was directly related to the number of free surface exposed basic groups whereas the maximum negative charge state was related to the number of free surface exposed acidic groups of the proteins. The surface exposed basic groups, which are involved in hydrogen bonding, have lower propensity to contribute to the positive charge of the protein. Similarly, the surface exposed acidic groups involved in salt bridges have lower propensity to contribute to the negative charge of the protein. Analysis of the crystal structure to determine the maximum charge state of protein in the electrospray mass spectrum was also used to interpret the reported mass spectra of several proteins. The results show that both the positive and the negative ion mass spectra of the proteins could be interpreted by simple consideration of the crystal structure of the folded proteins. Moreover, unfolding of the protein was shown to increase the positive charge-state because of the availability of larger number of free basic groups at the surface of the unfolded protein.

A Computational Model for Protein Ionization by Electrospray Based on Gas-Phase Basicity

“…Next, we propose a simple mathematical model based on the results of these atomistic investigations. This model has no adjustable parameters and it reproduces the well-known correlations between the protein net charge, q, and both mass [10] and solvent-exposed surface [17]. Our model hints to GB app as a key factor for the ionization of folded proteins, suggesting that protein ionization depends on intrinsic properties of the protein structure and on the GB of the solvent [4].…”

“…However, it might not necessarily hold for side chains. To [17] tackle this issue, the present study employs a combined MC/ MD sampling scheme using the OPLS/AA force field with GB corrections. Indeed, standard force fields for biomolecular simulations are unable to describe bond breaking and forming.…”

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

“…The two charge-state distributions may represent the following: conformation 1: A disordered state that when electrosprayed, yields more charging due to exposure of amino acid side chains, and less cation binding due to disordered binding structures. Indeed, Prakash and Mazumdar [81] showed that the higher charged states in positive ion ESI-MS are a consequence of unfolding, affording a greater number of surface basic groups. Denatured calmodulin also sprays with higher charge states than does the wild type [82].…”

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

“…Nevertheless, X-ray or NMR structures were not used to determine the exact number of salt bridges and their localization on the surface of the protein. This effort was later on made by Prakash and Mazumdar [40]. They showed that salt bridges and hydrogen bonds must be taken into account to determine the maximum charge state of protein in the positive and negative ion modes.…”