Basharat, Abdul RehmanXiong, XingzhaoXu, TianZang, YongSun, LiangliangLiu, Xiaowen2025-02-172025-02-172025Basharat AR, Xiong X, Xu T, Zang Y, Sun L, Liu X. TopDIA: A Software Tool for Top-Down Data-Independent Acquisition Proteomics. J Proteome Res. 2025;24(1):55-64. doi:10.1021/acs.jproteome.4c00293https://hdl.handle.net/1805/45722Top-down mass spectrometry is widely used for proteoform identification, characterization, and quantification owing to its ability to analyze intact proteoforms. In the past decade, top-down proteomics has been dominated by top-down data-dependent acquisition mass spectrometry (TD-DDA-MS), and top-down data-independent acquisition mass spectrometry (TD-DIA-MS) has not been well studied. While TD-DIA-MS produces complex multiplexed tandem mass spectrometry (MS/MS) spectra, which are challenging to confidently identify, it selects more precursor ions for MS/MS analysis and has the potential to increase proteoform identifications compared with TD-DDA-MS. Here we present TopDIA, the first software tool for proteoform identification by TD-DIA-MS. It generates demultiplexed pseudo MS/MS spectra from TD-DIA-MS data and then searches the pseudo MS/MS spectra against a protein sequence database for proteoform identification. We compared the performance of TD-DDA-MS and TD-DIA-MS using Escherichia coli K-12 MG1655 cells and demonstrated that TD-DIA-MS with TopDIA increased proteoform and protein identifications compared with TD-DDA-MS.en-USAttribution 4.0 InternationalData independent acquisitionMass spectrometryProteoform identificationTop-down proteomicsArticle