biostudies-literatureUnknownLe MTTwo-dimensional tandem mass spectrometry (2D MS/MS) provides in-depth biopolymer structural information previously not directly accessible with traditional one-dimensional MS/MS workflows, and in significantly less time (<1 second per sample). In this study, we enhance 2D MS/MS data analysis for greater applicability in omics workflows and address challenges in sequencing peptides in mixtures. We designed a graph-theory-based framework to efficiently manage, visualize, and maximize the structural information extractable from 2D MS/MS spectra. Graph analysis algorithms, including a PageRank-based method, are shown to deconvolve MS/MS signals and group together product ions from the same presursor peptide, enabling the reconstruction of peptide fragmentation trees. From this, MS <sup><i>n</iChemical sciencehttps://www.ebi.ac.uk/biostudies/studies/S-EPMC12418246biostudies-literatureFramework for &lt;i&gt;de novo&lt;/i&gt; sequencing of peptide mixtures &lt;i&gt;via&lt;/i&gt; network analysis and two-dimensional tandem mass spectrometry.PMC12418246Holden DTLe MTZhu YGleich DFCooks RGDziekonski ETfalseFramework for &lt;i&gt;de novo&lt;/i&gt; sequencing of peptide mixtures &lt;i&gt;via&lt;/i&gt; network analysis and two-dimensional tandem mass spectrometry.Two-dimensional tandem mass spectrometry (2D MS/MS) provides in-depth biopolymer structural information previously not directly accessible with traditional one-dimensional MS/MS workflows, and in significantly less time (<1 second per sample). In this study, we enhance 2D MS/MS data analysis for greater applicability in omics workflows and address challenges in sequencing peptides in mixtures. We designed a graph-theory-based framework to efficiently manage, visualize, and maximize the structural information extractable from 2D MS/MS spectra. Graph analysis algorithms, including a PageRank-based method, are shown to deconvolve MS/MS signals and group together product ions from the same presursor peptide, enabling the reconstruction of peptide fragmentation trees. From this, MS <sup><i>n</i2025-01-01T00:00:00Z2025 Sep2026-06-02T00:24:09.017Z2026-05-24T03:07:16.605ZS-EPMC124182464093307310.1039/d5sc03762j