TopDIA: A\nSoftware Tool for Top-Down Data-Independent\nAcquisition Proteomics

Top-down mass spectrometry is widely used for proteoform\nidentification,\ncharacterization, and quantification owing to its ability to analyze\nintact proteoforms. In the past decade, top-down proteomics has been\ndominated by top-down data-dependent acquisition mass spectrometry\n(TD-DDA-MS), and top-down data-independent acquisition mass spectrometry\n(TD-DIA-MS) has not been well studied. While TD-DIA-MS produces complex\nmultiplexed tandem mass spectrometry (MS/MS) spectra, which are challenging\nto confidently identify, it selects more precursor ions for MS/MS\nanalysis and has the potential to increase proteoform identifications\ncompared with TD-DDA-MS. Here we present TopDIA, the first software\ntool for proteoform identification by TD-DIA-MS. It generates demultiplexed\npseudo MS/MS spectra from TD-DIA-MS data and then searches the pseudo\nMS/MS spectra against a protein sequence database for proteoform identification.\nWe compared the performance of TD-DDA-MS and TD-DIA-MS using Escherichia coli K-12 MG1655 cells and demonstrated\nthat TD-DIA-MS with TopDIA increased proteoform and protein identifications\ncompared with TD-DDA-MS.