MULTI-LEVEL CHARACTERIZATION OF PROTEOFORM DIVERSITY AND POST-TRANSLATIONAL MODIFICATIONS VIA COMPLEMENTARY MASS SPECTROMETRIC STRATEGIES AND UNIPROT ANNOTATION

Abstract

The fusion of top-down and bottom-up mass spectrometry (MS) in tandem has gradually become a predominant method for revealing the whole picture in the case of proteoforms and post-translational modifications (PTM). Top-down MS is analyzing whole intact proteins which gives complete information about the different forms the material might be actually existing while bottom-up MS tears the proteins apart into peptides thus making the MS sensitive enough for the analysis along with specific site localization of the PTMs. The recent research has been showing that the combination of these two complementary approaches not only increases the coverage of the proteome but also improves the accuracy of PTM and validation through proteoforms. Tools such as PTM-TBA and ProteoCombiner make the integration of the two types of data from MS along with running the protein knowledge bases like UniProt for identifying, localizing, and validating the PTMs with even more confidence. The reviews by Allen and Eyers (2023) and others discuss the drawbacks of top-down workflows due to fragmentation efficiency, data complexity, and computational interpretation, while bottom-up methods are still not able to produce intact proteoforms. Hybrid approaches, as demonstrated by Wu et al. and later on through the development of computational pipelines, are able to deal with these problems since they make use of the strengths coming from both strategies. All together, these breakthroughs are proving the combination of top-down and bottom-up MS along with database annotation to be the most reliable method for precise characterization of proteoforms and thus deeper understanding of protein structure, regulation, and cellular function.