The use of electrospray ionization mass spectrometry in proteomics to identify different subunits in protein complexes is a growing and important technique to increase understanding of protein structure and function. As mass spectrometry is used more often for the identification of proteins in complexes, optimized software for making accurate assignments becomes more important. There are currently many options available for researchers in this field for data analysis. Massign is another tool available that may have greater success in the assignment of spectra.1
Morgner and Robinson report using Massign software to correctly identify a portion of ATPase from Enterococcus hirae, a protein complex of previously unknown composition. The algorithm was able to identify several different complexes of subunits and accurate ratios of subunits present. By being able to simultaneously measure mass, subunit composition, stoichiometry, and topology, Massign has the potential to be an important tool for the proteomics of protein complexes.
Many proteins in the cell exist in large complexes with other proteins. Understanding these complexes is an increasingly important problem for the field of proteomics. Several different kinds of information about protein complexes can be elucidated using mass-spectrometry techniques. In the case where the subunit composition of a complex is unknown, the identities of the proteins can be determined or a previously unknown protein or subunit may be identified. The ratios of the different proteins can be measured quantitatively to determine the stoichiometry of the complex subunits, or based on fragmentation of the complex, subunit or subcomplex interactions or overall topology of the complex can be determined.
Massign is a new software package designed to increase accuracy of assignment of mass-spectrometry data from large protein complexes.1 This new program accomplishes this task in a few different ways. First, by using an improved peak-fitting routine, more accurate measurements of masses of subcomplexes and better resolution of charge-state envelopes can be obtained. Second, by comparing gas and solution phase products of dissociation, differences in solvation can be determined for complexes and subcomplexes. Both of these approaches address the primary problems associated with mass spectra generated from large complexes: primarily, that the spectra are complicated. Complicated spectra is a product of multiple charge states in the proteins (often high-charge states) and overlapping mass envelopes masking lower abundance species that have mass to charge ratio in the same range as a more abundant complex. Complicated spectra can also arise from the presence of solvent molecules, cofactors, or posttranslational modifications made to different subunits in the complex, all of which might be unknown.
The improved peak-fitting routine employed by Massign consists partially of smoothing the peak series and establishing the charge state for subcomplexes, subunits, and the complex in the spectra and then assigning complexes to the different mass / charge species present in the spectra. Massign is able to assign charge states automatically or semiautomatically. It does this by selecting the top of the most abundant peak in a set of peaks and calculates the distance (in m/z) to the adjacent peaks for a theoretical species and compares it to the actual spectrum. The theoretical spectrum can be overlaid with the experimentally determined spectrum for each of the subcomplexes until the charge state is obtained and an accurate mass can be calculated.
Another feature of this program is the differentiation between subcomplexes formed by collision induced dissociation (CID) and subcomplexes formed in solution. The dissociation of subcomplexes can give clues to protein-protein interactions or topology of the overall complex. By analyzing complexes in both the solution and gas phase, subcomplexes loss through pairwise interaction can be mapped and compared to the data obtained through CID. CID can be used on different solution subcomplex species to further dissociate subunits into smaller subcomplexes in a MS/MS fashion. Eventually, the complex can be completely dissociated into subunits. Establishing these relationships between the complexes allows the software to rebuild hypothetical complexes that take into account the fragmentation / dissociation data.
Other software packages are also available for analysis of complexes from similar proteomics studies. Algorithms such as MaxQuant2 or SUMMIT3 have been used for assignment of subunit composition or topology of complexes. However, MaxQuant was developed for quantitative analysis of spectra and not optimized for whole proteins or larger protein complexes.2 Likewise, SUMMIT has limitations as well. SUMMIT maps protein interactions within the complex but does not analyze spectra the way Massign does. Massign has a variety of features that give it the potential to be an important proteomics tool in analyzing mass spectrometry data of large protein complexes.
1. Morgner, N. and Robinson, C.V. (2012) ‘Massign: An Assignment Strategy for Maximizing Information from the Mass Spectra of Heterogeneous Protein Assemblies‘, Analytical Chemistry, 84 (6), (pp. 2939-2948)
2. Cox, J. and Mann, M. (2008) ‘MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification‘, Nature Biotechnology, 26 (12), (pp. 1367-1372)
3. Taverner, T., et al. (2008) ‘Subunit Architecture of Intact Protein Complexes from Mass Spectrometry and Homology Modeling‘, Accounts of Chemical Research, 41 (5), (pp. 617-627)