Proteoform Identification Using Multiplexed Top‐Down Mass Spectra
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Top‐down mass spectrometry (TDMS) is the method of choice for analyzing intact proteoforms, as well as their posttranslational modifications and sequence variations. In top‐down tandem mass spectrometry (TD‐MS/MS) experiments, multiple proteoforms are often co‐fragmented, resulting in multiplexed TD‐MS/MS spectra. Due to their increased complexity, compared to spectra from single proteoforms, multiplexed TD‐MS/MS spectra present significant challenges for proteoform identification and quantification. Here we present TopMPI, a new computational tool specifically designed for the identification of multiplexed TD‐MS/MS spectra. Experimental results demonstrate that TopMPI substantially increases the sensitivity and accuracy of proteoform identification in multiplexed TD‐MS/MS spectral analysis compared to existing tools.
Summary
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Top‐down mass spectrometry (TDMS) is a powerful technique for analyzing intact proteoforms; however, identifying multiple co‐fragmented proteoforms from multiplexed tandem mass spectrometry (MS/MS) spectra remains a significant challenge.
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In this paper, we introduce TopMPI, a new computational tool specifically designed to identify multiplexed TD‐MS/MS spectra using a two‐round database search strategy.
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Compared to existing tools, TopMPI significantly improves the sensitivity and accuracy of proteoform identification from multiplexed MS/MS spectra. The development of TopMPI enhances the identification of low abundance proteoforms in complex biological samples and increases the potential of TDMS for discovering proteoform biomarkers in disease studies.
