Abstract
Top-down proteomics analyzes intact proteoforms with all of their post-translational modifications and genetic and RNA splice variants. In addition, modifications introduced either deliberately or inadvertently during sample preparation, that is, via oxidation, alkylation, or labeling reagents, or through the formation of noncovalent adducts (e.g., detergents) further increase the sample complexity. To facilitate the recognition of protein modifications introduced during top-down analysis, we developed MSTopDiff, a software tool with a graphical user interface written in Python, which allows one to detect protein modifications by calculating and visualizing mass differences in top-down data without the prerequisite of a database search. We demonstrate the successful application of MSTopDiff for the detection of artifacts originating from oxidation, formylation, overlabeling during isobaric labeling, and adduct formation with cations or sodium dodecyl sulfate. MSTopDiff offers several modes of data representation using deconvoluted MS1 or MS2 spectra. In addition to artificial modifications, the tool enables the visualization of biological modifications such as phosphorylation and acetylation. MSTopDiff provides an overview of the artificial and biological modifications in top-down proteomics samples, which makes it a valuable tool in quality control of standard workflows and for parameter evaluation during method development.
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