Recently, data-independent acquisition (DIA) has become a compelling alternative to DDA for proteomics analysis enabeling greater data completeness across samples 25, 26, 27, 28. Thus far, ubiquitinome studies have employed data-dependent acquisition (DDA) methods combined with label-free or isotope-based quantification 24. These requirements, which largely stem from the low stoichiometry of the modification, come at the expense of throughput, robustness, and quantitative accuracy. However, large-scale analysis of ubiquitination events to study key signaling components remains challenging since in-depth diGly proteome coverage requires relatively large sample amounts and extensive peptide fractionation. The commercialization of such antibodies has accelerated MS-based ubiquitinome analysis and enabled a variety of quantitative, systems-wide studies 19, 20, 21, 22, 23. A recently described antibody targets a longer remnant generated by LysC digestion to exclude ubiquitin-like modifications such as NEDD8 or ISG15 18, however, the contribution of diGly sites derived from ubiquitin-like modifications is very low (<6%) 15. Enrichment strategies employing such antibodies have enabled identification of thousands of ubiquitination sites by MS 15, 16, 17. After trypsinization previously ubiquitinated peptides bear a signature diGly remnant that can be targeted by a specific antibody 14. Early reports to catalog ubiquitin conjugated proteins from yeast and human described various enrichment methods including the use of epitope-tagged ubiquitin or ubiquitin-associated domains (UBA) 11, 12, 13. However, due to low stoichiometry of ubiquitination and varying ubiquitin-chain topologies, comprehensive profiling of endogenous ubiquitination is challenging and requires one or more enrichment steps prior to MS analysis 10. Protein ubiquitination is one of the most widely studied PTMs in the field of mass spectrometry (MS)-based proteomics. Deregulation of this highly complex process has been linked to numerous diseases including neurodegenerative diseases 3, 4, autoimmunity 5, 6, and inflammatory disorders 7, 8, 9. Ubiquitin itself can be ubiquitinated N-terminally or via one of its seven lysine residues, giving rise to a plethora of chain topologies, which encode a diverse and specific set of biological functions 1, 2. Its removal is mediated by an enzyme family called deubiquitinating enzymes (DUB). A ubiquitin conjugation cascade, involving ubiquitin activating (E1), conjugating (E2), and ligating (E3) enzymes, mediates the covalent attachment of the 76 amino acid long ubiquitin molecule to a ε-amine group of a lysine residue on a substrate protein. Ubiquitination is a reversible and highly versatile post-translational modification (PTM) involved in virtually all cellular processes. An in-depth, systems-wide investigation of ubiquitination across the circadian cycle uncovers hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting new connections between metabolism and circadian regulation. Applied to TNF signaling, the workflow comprehensively captures known sites while adding many novel ones. This approach identifies 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells – double the number and quantitative accuracy of data dependent acquisition. Here, we develop a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. Protein ubiquitination is involved in virtually all cellular processes.