Proteoform Identification Using Multiplexed Top‐Down Mass Spectra
Zhige Wang, Xingzhao Xiong, Xiaowen LiuABSTRACT
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
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. 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. 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.