Massign: an assignment strategy for maximizing information from the mass spectra of heterogeneous protein assemblies.

Electrospray ionization mass spectrometry (ESI-MS) has evolved into a powerful adjunct for structural biology, helping to unravel the quaternary structure of protein complexes. Increasing interest has led to the study of ever larger multicomponent systems. Investigating these large complexes with ESI has meant that progressively more complicated mass spectra have been recorded. Correct assignment of these spectra is essential to maximize the information content available. Here we present a new assignment strategy and a supporting software package that allows the investigation of large heterogeneous systems, previously beyond the scope of full spectral assignment due to their complexity. The strategy involves two parts. The first includes a peak fitting routine to determine charge state distributions and consequently the masses of the various subcomplexes. The second module distinguishes between solution and gas phase products depending on their mass to charge ratio and assigns these charge states to different subunit combinations. These fitting and assignment routines contain many internal checks for consistency and reveal mass shifts, dependent upon desolvation conditions and small molecule binding. Using a rotary ATPase as a working example, we show how this assignment strategy is capable of determining the stoichiometry and interactions of the 8 different subunits within this 29-subunit assembly.