Mapping the pathway of DIA innovation, from DDA to the SYNAPT XS

Author: Joanne Ballantyne

Evolving customer requirements have driven Waters’ innovation in data independent acquisition (DIA) over 20 years. Fueled by the needs of our customers, Waters’ legacy spans the commercialization of data dependent acquisition (DDA) to the development of our pioneering DIA technologies, including MSE, HDMSE and SONAR. The latest advances are now available side-by-side on the SYNAPT XS.

Since its commercialization in 1996, DDA has enabled researchers to determine molecular structures by fragmenting selected ions through tandem mass spectrometry. Though DDA has successfully been used to provide reliable identifications of molecules in heterogeneous mixtures, the limitations of the method for complex mixtures were quickly highlighted by the research community.

A variety of DIA methods have since been introduced as viable alternatives, which improve the depth of coverage by co-fragmenting all detectable precursor ions. This removes ion selection bias, allows simultaneous quantification and identification and is a molecular record of the experiment that can be re-interrogated many times with differing questions in mind. Waters has played a significant role in advancing DIA with the application of ion mobility as an additional component that separates ions based on their size, shape, charge and mass. For detailed information on the evolution of DIA innovation, read our white paper.  Here we further explore the latest advances in DIA and how the SYNAPT XS signifies a culmination of Waters’ leadership in DIA method development.

The Legacy of DIA (Data Independent Acquisition)

HDMSE and the benefits of ion mobility

Building on the success of MSE  1,2&3 in 2012, Waters collaborated with bioscience researchers to develop a new DIA methodology for enhancing the ability of liquid chromatography – mass spectrometry (LC-MS) to handle complex mixtures. The end result was HDMSE – ion mobility-enabled MSE – which supplies time-aligned fragmentation of ions separated in an ion mobility cell.

During an HDMSE acquisition, molecular fragmentation is performed after precursor ion mobility separation, and the drift time of the precursor and product ions can be used to correlate the two. Combining LC-MS and ion mobility separation produces a significant increase in peak capacity, making the methodology particularly suited to the investigation of complex mixtures.


The application of HDMSE ion mobility separation to LC-MS was first reported on bottom-up proteomic experiments in 2012, resulting in a 160% increase in the number of identified peptides and proteins observed through the use of ion mobility assisted acquisitions. [4]

Recent applications of HDMSE

Since the commercialization of HDMSE, the methodology has been widely applied to the analysis of complex mixtures. This year, LC–IMS–MS/MS technology, utilizing the HDMSE mode of data independent acquisition, was employed in a metabolomics research paper that won first prize in the Metabolomics Publications Awards in the category of Original Article. The paper discussed a method for rapid profiling of polar metabolites in rat urine, which resulted in an improvement in spectral quality by separating co-eluting metabolites, and provided a fourfold reduction in analysis time. [5] The results highlight the strong benefits of LC-MS combined with ion mobility separation for metabolomic studies where high-throughput, reproducible and reliable methodologies are needed.

The field of proteomics has also significantly advanced since the first application of HDMSE where ion mobility-assisted DIA mass spectrometry is now a commonly used tool for structural investigations. A recent study employed the method to evaluate the impact of hypoxia on the proteome of the prostate. [6] As tumor hypoxia is known to affect the responsiveness of cancer cells to chemotherapy and radiotherapy, the study investigated the impact of hypoxia on the proteome of the prostate and whether drugs that reduce hypoxia could have value as prostate cancer therapy. Hypoxia-induced alterations in the proteome of prostate cancer cells were measured using HDMSE and demonstrated that under hypoxic conditions, differentially expressed proteins appear to regulate structural and binding processes. This suggests a potential role for drugs that can negate the protective effect of hypoxia on prostate cancer cells and increase their susceptibility to therapy. 

Combining the power of HDMSE and SONAR

To extend the capabilities of DIA, Waters introduced a multiplexed strategy featuring a fast-scanning quadrupole – known as SONAR – which was revealed at ASMS 2016. The methodology utilizes a resolving quadrupole window that slides over a selected mass range during each MS scan. The ions are transmitted in sequence and precursor and fragment ion data are used to generate high quality fragmentation spectra. In 2017, MDs at Imperial College London collaborated with Waters to successfully apply SONAR to an investigation of lipidomic-based clinical questions relating to male fertility, which achieved a significantly greater depth of lipidomics analysis than previous studies. [7]


Both SONAR and HDMSE increase analytical peak capacity and provide scientists with ‘clean and clear’ fragmentation data, though based on different molecular properties. By offering the two modes of acquisition in a single platform, Waters’ SNYAPT XS has taken another leap forward by supplying a truly unique investigative toolbox for the in-depth interrogation of complex mixtures. This innovation means that by layering complementary fragment ion information for every detectable component, researchers are already benefiting from more unambiguous confirmation of compound identity.

Continuing to lead innovation in DIA

Since 1996, Waters has collaborated with scientists to introduce, develop and commercialize new modes of DIA that have revolutionized the way complex mixtures are mined and characterized. The work has culminated in the SYNAPT XS, which employs complementary DIA techniques to help scientists see more than ever before. By combining the power of two pioneering DIA methodologies, Waters is providing researchers with access to over two decades of innovation that will drive their research forward.

You can learn more about the ultimate flexibility of the SYNAPT XS here.


  1. Bateman et al. J Am Soc Mass Spectrom. 2002 Jul;13(7):792-803. doi: 10.1016/S1044-0305(02)00420-8.
  2. Silva et al. Anal Chem. 2005 Apr 1;77(7):2187-200. doi: 10.1021/ac048455k.
  3. Plumb et al. Rapid Commun Mass Spectrom. 2006;20(13):1989-94. doi: 10.1002/rcm.2550.
  4. Rodriguez-Suarez et al. Current Analytical Chemistry 2013; 9(2):199-211. doi:10.2174/157341113805218947.
  5. King et al. Metabolomics. 2019 Jan 22;15(2):17. doi: 10.1007/s11306-019-1474-9.
  6. Ross et al. Clin Chem Lab Med. 2020 Jun 25;58(6):980-993. doi: 10.1515/cclm-2019-062
  7. Gethings et al. Rapid Commun Mass Spectrom. 2017 Oct 15;31(19):1599-1606. doi: 10.1002/rcm.7941.