Data-independent acquisition (DIA) allows for unbiased, robust quantification over thousands of samples, ultimately providing deeper proteome coverage than data-dependent acquisition (DDA). In this article, we break down the advantages of DIA for accurate, robust, and reliable proteomics insights.
One of the key advantages of DIA is its ability to capture virtually all peptides present in a sample, providing a comprehensive view of the proteome. Unlike DDA, where specific ions are selected for fragmentation based on their intensity or abundance, DIA acquires all fragment ions within a predefined mass-to-charge (m/z) range systematically and impartially, minimizing missing values in the data.
Another advantage of DIA data is that it can be re-interrogated, without the need to repeat sample analysis. With DDA, any of the ions that weren’t selected for fragmentation are not stored, meaning re-interrogation is impossible. Re-interrogation of DIA data means interferences can be removed, and additional quantitative information can be obtained on proteins that were missed the first time round, ultimately improving the accuracy of the data.
DIA lacks selection bias and offers higher sensitivity, meaning even low-abundance peptides, which are often overlooked by DDA, can be detected. This is important as most biologically and disease-relevant proteins are found in the pg/ml range. While DDA’s coverage can be improved with deep fractionation, this introduces large overheads and experimental variability into the data.
Because DIA analyzes all fragment ions across a predefined mass-to-charge (m/z) range every run, there are minimal data gaps and missing values. Studies show DIA exhibits remarkable data completeness, reducing missing values to as low as 1.6%, compared to around 50% for DDA.
This high-quality, robust data means DIA is highly reproducible – by 2015, DIA was already outperforming DDA in terms of depth and quantification accuracy, boasting a reproducibility rate above 98%. This high reproducibility across multiple conditions, samples, and time points, makes DIA an excellent choice for large-scale proteomics studies.
Unlike other approaches, such as affinity-based assays, DIA-MS collects data in a completely unbiased way as all peptides are acquired independent of physiological characteristics, such as patient or disease status. In addition, DIA is very versatile and can even provide accurate identification and quantification of different isoforms, post-translational modifications, and cleavage products. This allows for hypothesis-free discovery and does not require prior knowledge of the protein composition of a sample, allowing researchers to explore uncharted territory with reproducible results.
As DIA offers unbiased and comprehensive coverage, it is the perfect choice for discovery proteomics. But DIA is not limited to just protein identification; it also enables absolute protein quantification, making it useful for targeted proteomics approaches.
Unlike PRM (parallel reaction monitoring) which targets specific peptides, DIA collects data from wide, evenly-spaced precursor isolation windows. SWATH (sequential window acquisition of all theoretical fragment ion spectra), a DIA method invented and developed by Sciex, ETH Zurich, and Biognosys in collaboration with proteomics pioneer Ruedi Aebersold, allows the identification of peptides from complete fragment ion maps acquired from a given sample. The data from these maps can then be mined in a targeted manner using spectral libraries, making DIA-MS the perfect choice for large-scale, label-free quantitative studies. Using stable isotope-labeled peptide internal standards (SIS) in DIA mode allows for absolute quantification by comparing the intensity of the SIS peptide peak with the peptide of interest’s peak. DIA makes this comparison reliable and accurate, enhancing reproducibility.
In summary, DIA offers comprehensive and deep coverage, reproducibility, versatility, and unbiased identification with unparalleled precision and accuracy. Interested in using DIA? Biognosys’ Spectronaut® software performs DIA data analysis with cutting-edge algorithms that can characterize thousands of proteins. Learn more about our recent work using Spectronaut for DIA analysis below.