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A Decade of DIA and Key Milestones: The 10-Year Anniversary of SWATH DIA

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“DIA is THE method for LCMS based proteomics,” writes Dr. Benjamin Orsburn of Johns Hopkins University as one of his key takeaways after attending the 2022 World HUPO congress in Cancun, Mexico. This comes as no surprise to the community as SWATH DIA (data-independent acquisition) mass spectrometry (MS) has surpassed the expectations of the proteomics research community over the past decade. Having just celebrated its 10-year anniversary, SWATH DIA is a next-generation liquid chromatography tandem mass spectrometry (LC-MS/MS) approach that is quickly proving to be the acquisition method of choice among proteomics researchers. Particularly in omics-based research, the efforts to advance SWATH DIA have enabled trending areas of research, such as personalized medicine and the rise of single-cell research.  


Data-dependent acquisition (DDA) and selected reaction monitoring (SRM) are two LC-MS/MS techniques that traditionally have been used to detect analytes in a sample. However, compared to these predecessors, SWATH DIA is a more sensitive measure for identifying analytes and other relevant data points in an analysis. In a SWATH DIA method, the full mass-to-charge ratio (m/z) range of the sample of interest is divided into numerous windows, where all ions within each window are fragmented. This results in in-depth identification and accurate peptide quantification without being limited to profiling predefined peptides of interest. “SWATH [DIA] is a powerful method to assist researchers to develop quantitative methods in a rapid manner, allowing the measurement of all compounds within a sample in a targeted manner. It bridges the gap between DDA methods and MRM, providing rapid identification and quantification of compounds with MRM qualities,” explains Stephen Tate, PhD, of SCIEX, one of the key developers behind SWATH DIA.


Key milestones for SWATH DIA


The 10-year anniversary of SWATH DIA signifies a period of immense growth that is only expected to continue to evolve, influencing a wide range of fields and precision medicine. While 10 years ago only roughly 10% of a human tissue proteome could be quantified, today the proteomics community can quantify quasi-complete tissue proteomes using SWATH DIA. This dramatic improvement is the result of better sample preparation methods, advanced chromatography, refined acquisition methods, more efficient mass spectrometers and advanced data processing algorithms. As we look forward to the future innovations this technique will bring, we cannot forget the revolutionary key milestones SWATH DIA has helped researchers achieve.


Initiating the discovery of new proteins


Ruedi Aebersold, PhD, the retired head of the Department of Biology at ETH Zurich, has significantly contributed to the creation and advancement of SWATH DIA together with his lab and the research team at SCIEX. From 2011 until the present day, his efforts – along with the contributions of many other distinguished researchers – put SWATH DIA on the map for researchers to access. “SWATH [DIA] is ideal for proteomics, allowing us to investigate, for example, how genomic variability is translated into phenotypes, and go on to perform biomarker studies,” states Aebersold. “For the first time, we can measure hundreds of similar samples reliably and with moderate effort, cells of exactly the same type but at different states, samples from a large number of individuals, or from different tissues from one individual, and make meaningful comparisons,” he continues. In fact, while the first implementation of SWATH DIA was on a QTOF system, the workflow has become more universal than originally expected, and is being used with orbitrap-based instruments and even with ion mobility separation technologies. 


Lukas Reiter, PhD, the chief technology officer at Biognosys, has worked closely with Aebersold and the research and development team at SCIEX to develop this technology, having just moved from Aebersold’s group to the startup at the time. During its early stages of development, the analysis of SWATH DIA data was “daunting and results typically meager,” explains Reiter. He adds that it was after some discussions with the Aebersold group members, Pedro Navarro, PhD, and Ludovic Gillet, PhD, that a eureka moment occurred: they could apply a recently developed algorithm with an intermediate targeted extraction process that would convert the SWATH DIA data into data that looked like MRM. Reiter describes SWATH DIA as the “combination of seamless data acquisition and the robust data processing that result in consistent and precise quantification.”


One of the most extraordinary achievements in this field comes from the HUPO Human Proteome Project, which recently published that over 90% of human proteins are detectable. Proteins classified as “missing” were also identified, contributing to knowledge bases that inform the development of life-saving medicines. Similarly, the CHO spectral library for SWATH DIA has quantitatively profiled over 10,000 proteins related to Chinese hamster ovary cell lines. Not only do projects like HUPO contribute to the overall success of many proteomics projects, but they also improve standards for data-sharing, quality assurance and future guidelines for protein discovery.


Enhancing clinical lab capabilities and accelerating development in oncology and neuroscience research


As SWATH DIA becomes the leading technique for mass spectrometry research, fields such as oncology are taking advantage of its new possibilities. The number of applications in these fields has increased significantly, including molecular classification, oncogenic pathway analysis, drug and biomarker discovery, and unraveling mechanisms of therapy response and resistance.


An example of this is the Stoller Center on Cancer, a facility dedicated to understanding cancer biomarkers and developing more personalized and precise treatment options. The Center benefits from SWATH DIA in multiple ways. Currently, they are using it to inform new treatments for chronic myeloid leukemia, potentially extending the lives of patients diagnosed with the condition. The Stoller Center, along with other centers and clinical labs like it, uses SWATH DIA to perform proteomic analyses at a higher bandwidth than ever before, increasing the number of large-scale projects they can work on at once.

SWATH DIA has also proved to be a successful methodology in neuroscience, unveiling new information about proteins significant to diagnosing and treating Alzheimer’s disease. In a
study of over 3,200 older adults that featured SWATH DIA as the primary methodology, Basisty and assisting researchers found that certain senescence factors – secretions from senescent cells – elevated in plasma during the aging process. This discovery contributes to a growing effort to identify biomarkers relevant to signs that indicate the presence of Alzheimer’s disease pathology in the brain and other parts of the body.


Responding to the COVID-19 pandemic


Markus Ralser, PhD, is a professor and the head of the Biochemistry Institute at Charité University Medicine, who has become instrumental in the advancements of SWATH DIA. Furthermore, his work provides evidence of the global impact SWATH DIA has on research. Most recently, his team used SWATH DIA (and short-gradient, high-flow liquid chromatography) to identify over 25 clinical classifiers, also known as biomarkers, that are associated with mild to severe strains of COVID-19. The SWATH DIA methodology is described as a “rapid-response, clinically actionable technology adaptable to infectious outbreaks,” speaking to its versatility.


Paving the way for future research


Improving SWATH DIA methodologies is key to paving the way for future research. Earlier this year, SCIEX launched Zeno SWATH DIA, which was developed to address sensitivity and speed issues that were noticed in previous versions. Not only will this allow researchers to gather more meaningful, unbiased data, but it will also reduce the time it takes to collect it – shortening the data acquisition and analysis process.


Like most new technologies, the developmental process is constantly evolving. The speed and quality of Zeno SWATH DIA will undoubtedly continue to improve, creating more efficient processes. It is evident that DIA is a central catalyst for shifting mass spectrometry from a discovery science to one that embraces clinical trials and research involving more than one sample. This updated method allows for easily reproducible methodologies, furthering scientists’ ability to analyze new topics. 


With over 2,400 citations documented in the past decade, it is also clear that the original DIA manuscript continues to make a profound impact on proteomics research. Only 10 years after its introduction, it is safe to say that SWATH DIA is the future of MS technology. The questions that remain are: Where do we go from here? What are the next steps to accelerate DIA further?


New technologies such as Zeno SWATH DIA can help propel SWATH DIA into the future, enabling those in the life sciences community to continue developing groundbreaking research.



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