Metastable ion formation and disparate charge separation in the gas-phase dissection of protein assemblies studied by orthogonal time-of-flight mass spectrometry

Abstract

The dissection of specific and nonspecific protein complexes in the gas phase is studied by collisionally activated decomposition. In particular, the gas phase dissection of multiple protonated homodimeric Human Galectin I, E. Coli Glyoxalase I, horse heart cytochrome c, and Hen egg Lysozyme have been investigated. Both the Human Galectin I and E. Coli Glyoxalase I enzymes are biologically active as a dimer, exhibiting molecular weights of approximately 30 kDa. Cytochrome c and Lysozyme are monomers, but may aggregate to some extent at high protein concentrations. The gas phase dissociation of these multiple protonated dimer assemblies does lead to the formation of monomers. The charge distribution over the two concomitant monomers following the dissociation of these multiple protonated dimers is found to be highly dissimilar. There is no evident correlation between the solution phase stability of the dimeric proteins and their gas-phase dissociation pattern. Additionally, in the collisionally activated decomposition spectra diffuse ion signals are observed, which are attributed to monomer ions formed via slow decay of the collisionally activated dimer ions inside the reflectron time-of-flight. Although, the formation of these diffuse metastable ions may complicate the interpretation of collisionally activated decomposition mass spectra, especially when studying noncovalent protein complexes, a simple mathematical equation may be used to reveal their origin and pathway of formation.

Thermal dissociation of the protein homodimer ecotin in the gas phase

“…Consequently, the general observation of highly asymmetric monomer charge states in the CID spectra of protein dimers [4,5] may result, in part, from the dissociation of asymmetric charge isomers produced by the ES (nanoES) process. It is likewise possible that gaseous multimeric protein complexes produced by ES also consist of multiple charge isomers.…”

“…The magnitude of the repulsion will depend on the total number of charges, their distribution, and location on the proteins. For a homodimer such as ecotin, electrostatic repulsion between the monomers is expected to be greatest when the charge is evenly shared between the monomers, assuming that the monomers have similar conformations [4,6]. Secondly, repulsion within each monomer can induce partial or complete unfolding of the monomers, with higher monomer charge states leading to a greater degree of unfolding.…”

“…The use of multiple stages of MS with ion activation/dissociation (MS n ) to dissect gaseous assemblies into their constituent subunits represents an attractive, yet unproven, strategy for determining subunit composition and topology. In recent years, a number of MS n studies of gaseous protein dimers [4,5], tetramers [6], and pentamers [7] have been reported. Homo-and heterodimers are readily decomposed into their individual subunits, most generally by collision-induced dissociation (CID).…”

“…An interesting feature of the dissociation behavior of the protein-protein complexes is the tendency for uneven sharing of charge between subunits, even in the case of homodimers. For example, the decomposition of four homodimers (human galectin I, E. coli glyoxalase I, horse heart cytochrome c, and hen egg lysozyme) yields a highly asymmetric charge distribution, with one of the subunits retaining as much as 73% of the dimer ion charge [4]. Even more pronounced charge state asymmetry is observed in CID experiments performed on cytochrome c-cytochrome b 5 complexes [5].…”

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

“…One challenge in using mass spectrometry for the general analysis of protein complexes is understanding the dissociation mechanism of these complexes in the gas phase. Many groups [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] have reported asymmetric dissociation behaviour for large multimeric proteins. A small subunit, typically a protein monomer, is ejected from the complex during dissociation, with the monomer carrying away a disproportionate amount of charge for its relative mass.…”