Evaluation of top-down mass spectral identification with homologous protein sequences

Abstract

Background: Top-down mass spectrometry has unique advantages in identifying proteoforms with multiple post-translational modifications and/or unknown alterations. Most software tools in this area search top-down mass spectra against a protein sequence database for proteoform identification. When the species studied in a mass spectrometry experiment lacks its proteome sequence database, a homologous protein sequence database can be used for proteoform identification. The accuracy of homologous protein sequences affects the sensitivity of proteoform identification and the accuracy of mass shift localization. Results: We tested TopPIC, a commonly used software tool for top-down mass spectral identification, on a top-down mass spectrometry data set of Escherichia coli K12 MG1655, and evaluated its performance using an Escherichia coli K12 MG1655 proteome database and a homologous protein database. The number of identified spectra with the homologous database was about half of that with the Escherichia coli K12 MG1655 database. We also tested TopPIC on a top-down mass spectrometry data set of human MCF-7 cells and obtained similar results. Conclusions: Experimental results demonstrated that TopPIC is capable of identifying many proteoform spectrum matches and localizing unknown alterations using homologous protein sequences containing no more than 2 mutations.