Principles, Applications, and Comparative Workflow of DIA and DDA

Principles of DIA

Data-Independent Acquisition (DIA) operates on the principle of comprehensive data acquisition across the entire mass range of interest. Unlike traditional methods, DIA does not rely on precursor ion selection. Instead, it systematically fragments all ions within predefined mass windows, generating a rich dataset that encompasses a broad spectrum of molecular species. This unbiased approach is particularly advantageous for identifying low-abundance molecules and provides a holistic view of the sample composition.

DIA's Role and Applications

Comprehensive Data Acquisition:

DIA excels in capturing a comprehensive range of ions in a single experiment, providing a snapshot of the entire sample composition. This characteristic is particularly valuable in complex samples, such as biological tissues or environmental matrices.

Unbiased Analysis:

The unbiased nature of DIA makes it an ideal choice for discovering novel molecules or elucidating unexpected components in a sample. This has profound implications in fields like metabolomics and proteomics.

Quantitative Analysis:

DIA facilitates accurate and reproducible quantification by acquiring data across the entire mass range, enabling the quantification of target analytes with high sensitivity and precision.

Principles of DDA

Data-Dependent Acquisition (DDA) is a more targeted approach. It selectively isolates and fragments precursor ions based on predefined criteria, often intensity or abundance. This targeted fragmentation allows for the identification and quantification of specific ions, offering a more focused analysis. However, the limitation lies in the potential omission of low-abundance or less intense ions, leading to a narrower scope of information compared to DIA.

DDA's Role and Applications

Targeted Ion Selection:

DDA's strength lies in its ability to selectively choose precursor ions for fragmentation based on predetermined criteria. This targeted approach is advantageous when specific ions need in-depth characterization.

Identification of Known Compounds:

DDA is well-suited for the identification of known compounds, making it a preferred method in fields where the focus is on targeted analysis rather than comprehensive profiling.

Dynamic Range:

DDA can handle a wide dynamic range of ion intensities, allowing for effective analysis in samples with highly variable concentrations of analytes.

Comparison of Full-Scan, Data-Dependent, and Data-Independent Acquisition Modes in Liquid Chromatography–Mass Spectrometry

Comparison of Full-Scan, Data-Dependent, and Data-Independent Acquisition Modes in Liquid Chromatography–Mass Spectrometry (Guo et al., 2020)

Comparative Workflow Analysis

The efficacy of any mass spectrometry methodology is intricately linked to its workflow – the series of steps from sample preparation to data analysis. In comparing the workflows of DIA and DDA, we gain valuable insights into their distinctive approaches and operational nuances.

Sample Preparation

DIA: DIA workflows typically start with comprehensive sample preparation, where the emphasis is on extracting and preparing all components of interest. The unbiased nature of DIA allows for the inclusion of a wide range of molecular species in the sample preparation process.

DDA: In contrast, DDA workflows often involve a more targeted sample preparation approach. Precursor ion selection criteria are predefined, guiding the extraction and preparation of specific molecular components. This targeted approach streamlines the sample preparation process.

Ionization and Mass Spectrometry

DIA: Once the sample is prepared, DIA employs a systematic approach to ionization and mass spectrometry. The mass spectrometer systematically fragments ions across predefined mass windows, ensuring comprehensive coverage of the mass spectrum. This systematic fragmentation is a key feature that sets DIA apart.

DDA: DDA, on the other hand, involves selective ionization and mass spectrometry. The mass spectrometer isolates and fragments precursor ions based on predefined criteria, often influenced by ion intensity or abundance. This targeted approach allows for in-depth characterization of specific ions.

Data Acquisition

DIA: DIA excels in simultaneous acquisition across the entire mass range. The resulting data set is comprehensive and unbiased, capturing a broad spectrum of molecular species. The approach minimizes the risk of missing low-abundance or unexpected analytes.

DDA: In DDA, the data acquisition process is more selective. Precursor ions meeting predefined criteria are chosen for fragmentation, leading to a dataset that is focused on specific molecular components. While this targeted approach provides detailed information about chosen ions, it may overlook less intense species.

Data Analysis

DIA: The comprehensive data set generated by DIA requires sophisticated data analysis tools capable of handling large volumes of information. Bioinformatics tools and algorithms designed for unbiased data interpretation are essential for extracting meaningful insights from DIA experiments.

DDA: DDA data analysis tends to be more straightforward, focusing on the interpretation of targeted ion data. However, challenges may arise in situations where unexpected or low-abundance ions become crucial, as they may be overlooked in the selective data acquisition process.

Data Acquisition Strategies

The heart of mass spectrometry lies in the acquisition of data, and understanding the strategies employed by Data-Independent Acquisition (DIA) and Data-Dependent Acquisition (DDA) is pivotal for unraveling their unique capabilities.

DIA Data Acquisition Strategies

  • Comprehensive Spectrum Coverage:

DIA adopts a holistic approach by systematically fragmenting all ions within predefined mass windows. This strategy ensures the acquisition of a comprehensive spectrum across the entire mass range of interest. The unbiased nature of DIA enables the detection of low-abundance and unexpected molecular species, contributing to a more thorough analysis.

  • Parallel Fragmentation:

One of the distinctive features of DIA is its ability to fragment multiple ions simultaneously. This parallel fragmentation enhances the efficiency of data acquisition, allowing for a more rapid and comprehensive coverage of the sample, which is particularly advantageous in high-throughput applications.

  • Data-Independent Ion Mobility:

In addition to mass information, DIA can incorporate ion mobility spectrometry, providing an extra dimension to separate ions based on their size and shape. This extension enhances the resolution and specificity of DIA data, contributing to a more detailed characterization of complex samples.

DDA Data Acquisition Strategies

  • Targeted Ion Selection:

DDA operates on a targeted strategy, selectively isolating and fragmenting precursor ions based on predefined criteria, often influenced by ion intensity or abundance. This targeted ion selection approach allows for in-depth characterization of specific molecular components, making DDA particularly suitable for the analysis of known compounds.

  • Priority-Based Fragmentation:

In situations where multiple precursor ions are present, DDA employs a priority-based strategy, focusing on the most abundant ions first. While this enhances the efficiency of data acquisition, it may lead to the exclusion of less intense ions, potentially limiting the scope of the analysis.

  • Dynamic Exclusion:

DDA often incorporates a dynamic exclusion feature, temporarily excluding previously selected ions from further fragmentation. This strategy aims to increase the diversity of ions selected for fragmentation, preventing over-repetition of the same ions and providing a more comprehensive view of the sample.

Comparative Analysis of Data Acquisition Strategies

In comparing the data acquisition strategies of DIA and DDA, DIA stands out for its unbiased and comprehensive approach, ensuring a broad spectrum of information. The simultaneous fragmentation of multiple ions and the potential incorporation of ion mobility contribute to its versatility. On the other hand, DDA's targeted strategy excels in in-depth characterization of specific ions, making it particularly valuable for targeted analysis.

We leverage a range of cutting-edge DIA technologies to enhance mass spectrometry capabilities. Our primary DIA technologies include SWATH-MS, SRM™, MSX-DIA, PCT-DIA and GPF-DIA. Each technology offers unique advantages, contributing to comprehensive and precise analyses in diverse experimental contexts.


Guo, Jian, and Tao Huan. "Comparison of full-scan, data-dependent, and data-independent acquisition modes in liquid chromatography–mass spectrometry based untargeted metabolomics." Analytical Chemistry 92.12 (2020): 8072-8080.

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