Resolving stoichiometries and oligomeric states of glutamate synthase protein complexes with curve fitting and simulation of electrospray mass spectra.

A complicating factor in analyzing electrospray ionization mass spectra of intact macromolecular heterogeneous protein complexes is the potential overlap of ions from different species present in solution. Therefore, it is often not possible to assign all ion signals. With the aim of allowing the more efficient and comprehensive analysis of very complex mass spectra of intact heterogeneous protein complexes we developed a software program: SOMMS. The program uses simple user input parameters together with Gaussian curve fitting to simulate putative mass spectra of protein (sub)complexes within a specified charge state window. In addition, the program can simulate spectra for heterogeneous protein complexes using bi- and multinomial distributions and it can calculate zero-charge spectra and relatively quantify the abundance of each component in a mixture. As a proof of concept we analyzed the complex mass spectra of alpha-glutamate synthase and alphabeta-glutamate synthase from Azosprillum brasilense. Using our program we could determine that alpha-glutamate synthase is in equilibrium between its dimeric, tetrameric, hexameric and dodecameric conformation, whereas alphabeta-glutamate synthase forms up to 15 different heterooligomeric assemblies composed of alpha- and beta-subunits. Thus, SOMMS allows resolving stoichiometries and oligomeric states of protein complexes even from very complicated mass spectra. These complexes could not be assigned by using maximum entropy calculations. We compared our mass spectrometry data on glutamate synthases with available X-ray, small-angle X-ray scattering and size-exclusion chromatography data.