DIA/SWATH-Based Quantitative PTM Analysis
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Experimental spectral libraries: Custom libraries are constructed by acquiring fragment ion spectra of modified peptides through preliminary data-dependent acquisition (DDA) experiments, yielding highly targeted and representative libraries.
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Predicted spectral libraries: Leveraging machine learning models such as Prosit or DIA-NN, predicted MS/MS spectra are generated from peptide sequences, enabling rapid development of large-scale, high-coverage libraries for PTMs.
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Site-specific differential analysis: Identification of modification sites that exhibit significant changes under varying experimental conditions;
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Construction of modification networks: Investigation of the functional roles of specific PTM types within cellular signaling pathways and transcriptional regulation mechanisms;
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Analysis of PTM crosstalk: Exploration of cooperative or antagonistic interactions among distinct PTMs, such as phosphorylation, acetylation, and ubiquitination.
Post-translational modifications (PTMs) play a central role in regulating cellular functions, signal transduction, metabolic processes, and the development of diseases. Modifications such as phosphorylation, acetylation, ubiquitination, and methylation confer spatiotemporal specificity and functional diversity to proteins, providing critical insights into their functional states. With advances in mass spectrometry, data-independent acquisition (DIA) techniques—particularly the SWATH-MS (Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) strategy—have emerged as powerful tools for quantitative PTM analysis. Characterized by high throughput, excellent reproducibility, and minimal bias, DIA/SWATH enables systematic profiling of modified proteins in complex biological samples.
Complexity and Challenges of Post-Translational Modifications
PTMs exhibit considerable complexity. Multiple modification types may occur on a single protein or even on the same amino acid residue, resulting in either synergistic or antagonistic regulatory effects. Furthermore, PTMs are typically present at low abundance, exhibit rapid dynamics, and are profoundly influenced by cellular conditions, external stimuli, and developmental stages. The structural heterogeneity of modification sites also imposes high demands on analytical resolution. Conventional approaches often suffer from incomplete data acquisition and low sample reproducibility, limiting their capacity for systematic analysis. DIA/SWATH-based mass spectrometry, with its comprehensive coverage and accurate quantification capabilities, offers a promising solution to these challenges.
DIA/SWATH-MS: Enabling High-Dimensional PTM Proteomics
The core principle of DIA/SWATH technology involves dividing the full mass-to-charge (m/z) scan range into multiple windows, systematically fragmenting all precursor ions within each window, and acquiring complete MS/MS spectra. This acquisition mode does not depend on precursor ion intensity and can capture low-abundance signals without selection bias, thereby facilitating unbiased and comprehensive analysis of all detectable peptides in a single run.
In PTM research, this strategy offers three major advantages:
1. Comprehensive and Consistent Data Acquisition
DIA/SWATH ensures that all samples are analyzed under identical conditions with consistent data dimensionality, significantly enhancing inter-batch comparability and data stability. This is especially beneficial for studies involving dynamic PTM events, such as drug treatment, cell cycle progression, or inflammatory responses.
2. Site-Specific Quantification of Modifications
Accurate localization of PTM sites is essential for meaningful biological interpretation. The high coverage of DIA data, combined with curated spectral libraries or advanced prediction algorithms, enables confident assignment of modification sites, allowing a transition from peptide-level to site-level PTM analysis.
3. Suitability for Complex Samples and Multi-Condition Studies
DIA/SWATH supports large-scale experimental designs involving multiple treatment conditions and sample groups, offering robust inter-batch consistency and high-throughput processing. These attributes make it particularly well-suited for multivariate statistical analyses, time-series modeling, and systems biology network construction.
Key Procedures of DIA in Post-Translational Modification Studies
The effective utilization of DIA/SWATH technology relies on a fully optimized and interdependent workflow, spanning from sample preparation to data interpretation, where each step critically supports the next.
1. Specific Enrichment of Modified Peptides
Post-translational modifications (PTMs) are typically characterized by low abundance, transient stability, and susceptibility to environmental fluctuations. To improve their detectability in mass spectrometry, enrichment strategies serve as the initial selection barrier. For instance, phosphorylation is commonly enriched using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO₂), whereas acetylation and ubiquitination are typically captured through immunoaffinity enrichment with modification-specific antibodies. Achieving high selectivity and minimizing non-specific binding are essential prerequisites for reliable DIA-based analysis.
2. Construction of DIA Spectral Libraries and Integration of Modification Information
In DIA workflows, spectral libraries represent a critical component for accurate identification and quantification of modified peptides. These libraries can be generated through two primary approaches:
High-quality spectral libraries substantially enhance the sensitivity and quantification accuracy of modified peptides and are particularly valuable for identifying novel or previously uncharacterized modification sites.
3. Data Analysis and Biological Interpretation
PTM data analysis encompasses not only quantitative assessment but also the elucidation of biological relevance. Key analytical focuses include:
At present, several DIA-compatible software platforms—including Spectronaut, DIA-NN, and Skyline—offer robust support for PTM analysis, facilitating the extraction of biologically meaningful insights from complex proteomic datasets.
DIA/SWATH-based quantitative PTM analysis enables robust and reproducible identification and quantification of modification sites across large-scale sample sets, thereby advancing the field from structural characterization to functional elucidation. MtoZ Biolabs is dedicated to providing high-quality SWATH-based quantitative proteomics services, supporting the investigation of intricate regulatory mechanisms of protein modifications and contributing to the expansion of life science frontiers.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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