Application of DIA Technology in Ubiquitin Proteomics
Ubiquitination is a major post-translational modification (PTM) in which the 76-amino-acid ubiquitin protein is covalently attached to lysine residues of substrate proteins. This modification regulates a wide range of cellular processes, including protein stability, signal transduction, and DNA repair. Conventional mass spectrometry strategies such as data-dependent acquisition (DDA) have enabled the identification of ubiquitinated peptides, yet they often suffer from sampling bias and missing values. In recent years, Data-Independent Acquisition (DIA), when coupled with diGly (K-ε-GG)-specific antibody enrichment, has enabled high-throughput and quantitative profiling of the ubiquitinated proteome. As a result, DIA has become an indispensable approach in ubiquitinomics, particularly for detecting low-abundance ubiquitination events, improving comparability across samples, and supporting large-scale functional screening experiments.
Reasons for Combining Ubiquitination with Data-Independent Acquisition (DIA) Technology
1. Comprehensive and Less Biased Acquisition to Improve Data Completeness
(1) DIA fragments all precursor ions within predefined m/z windows, enabling systematic acquisition with minimal stochastic sampling.
(2) Unlike DDA, which preferentially selects high-abundance peptides, DIA markedly reduces acquisition bias, facilitates the detection of low-abundance ubiquitinated peptides, and improves experimental reproducibility.
2. Improved Reproducibility and Quantitative Accuracy
(1) DIA typically provides more consistent quantitative coverage, with fewer missing values across samples. The coefficient of variation (CV) is often lower than that achieved by DDA.
(2) This is particularly important for comparing diseased versus normal states and for screening potential deubiquitinase (DUB) or E3 ligase targets.
Experimental Workflow of DIA-Ubiquitinomics
1. Sample Preparation Sample Preparation
(1) Cells or tissues are lysed and digested with trypsin.
(2) Ubiquitinated peptides carrying the diglycine remnant on lysine are then selectively enriched using anti-diGly antibodies, which constitutes the central step in DIA-based ubiquitinomics.
2. Spectral Library Building Spectral Library Building
(1) To improve peptide identification and matching efficiency, a large-scale spectral library is commonly generated using DDA measurements or in silico prediction.
(2) For example, Hansen et al. constructed a spectral library containing >90,000 diGly peptides, enabling the identification of up to ~35,000 diGly sites in a single DIA run.
3. DIA-MS Acquisition
(1) DIA acquisition is performed on high-resolution mass spectrometers (e.g., Orbitrap platforms), often using variable isolation windows.
(2) This strategy promotes broad coverage of ubiquitination-related signals and improves quantitative linearity.
(3) Data can be processed using dedicated DIA software such as Spectronaut or DIA-NN to enable high-throughput reporting.
4. Data Processing and Quantitative Analysis
(1) In addition to conventional library-based matching, library-free workflows (e.g., directDIA) can also be applied.
(2) This option is particularly advantageous when sample amounts are limited or when spectral libraries are incomplete. For instance, it has been reported in single-cell DIA applications.
Typical Research Case Demonstrations
1. Environmental Response and Signaling Pathway Applications
(1) Hansen et al. used DIA to profile ubiquitination sites within the TNF signaling pathway and identified a large number of previously unreported targets.
(2) These data further supported a role of ubiquitination in regulating intrinsic temporal dynamics of rhythm-associated proteins.
2. Mining Drug Mechanisms of Action
(1) Steger and colleagues used DIA to quantify >70,000 ubiquitination sites.
(2) They delineated rapid targets of USP7 inhibitors, observing extensive ubiquitination changes within as little as 2 minutes.
3. Single-Cell Level Ubiquitinomics
(1) Recent single-cell DIA studies have demonstrated the ability to identify hundreds to thousands of ubiquitinated peptides from a single cell or a small number of cells.
(2) This progress is expected to accelerate applications of ubiquitinomics in cellular heterogeneity, the tumor microenvironment, and related fields.
Summary of Advantages of DIA-Ubiquitinomics
Challenges of Combining DIA Technology with Ubiquitin Proteomics
1. Cost and Quality Control of Spectral Library Construction
(1) Large-scale spectral libraries often rely on extensive DDA runs, which increase cost and demand additional laboratory resources.
(2) Library quality directly influences the depth and reliability of DIA results.
2. Difficulty in Chain-Type Identification
Although DIA improves site-level coverage, accurate identification and quantification of polyubiquitin chain linkage types still typically require complementary approaches (e.g., neutral loss–based strategies or chemical probes).
3. High Barrier for Data Analysis
(1) DIA data are complex and often require specialized computational platforms such as Spectronaut or DIA-NN.
(2) High-performance computing resources and analytical expertise are critical to ensure reliable outputs.
Prospects for the Combination of Dia Technology and Ubiquitin Proteomics
1. Broader adoption and further standardization of single-cell ubiquitinomics.
2. Joint quantitative profiling of dual modifications (e.g., co-existing PTMs).
3. Deeper integration of artificial intelligence algorithms with in silico spectral libraries.
4. Expanded compatibility with clinical specimens and scaling to high-throughput platforms.
Advantages of MtoZ Biolabs in DIA ubiquitinomics
MtoZ Biolabs has established a mature DIA-Ubiquitinomics platform:
1. Optimized Antibody Enrichment Workflows: Efficient extraction of target diGly peptides from both cell and tissue samples.
2. Customized Spectral Library Construction: Project-level libraries supported by combined DDA and in silico–based strategies.
3. Dedicated Bioinformatics Team: Integrated interpretation reports powered by tools such as Spectronaut and DIA-NN.
4. Domain-Focused Support: Including circadian biology, biopharmaceutical R&D, single-cell analysis, and drug mechanism studies.
DIA has become a key methodological route for ubiquitination proteomics. If you plan to apply DIA to investigate cell-cycle regulation, disease mechanisms, or drug modes of action, MtoZ Biolabs can provide end-to-end customized support, from sample preparation to data analysis, to facilitate robust and high-impact discoveries. For additional details on service specifications, technical roadmaps, and collaboration options, please contact the MtoZ Biolabs expert team. We are committed to supporting and safeguarding your research projects.
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