Top-down mass spectrometry is widely used for proteoform identification, characterization, and quantification owing to its ability to analyze intact proteoforms. In the last 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 K-12 MG1655 cells and demonstrated that TD-DIA-MS with TopDIA increased proteoform and protein identifications compared with TD-DDA-MS.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11030422 | PMC |
| http://dx.doi.org/10.1101/2024.04.05.588302 | DOI Listing |
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