SWATH DIA (data independent acquisition)

DDA (also known as information dependent acquisition, or IDA) is one of the most well established accurate mass acquisition strategies. With DDA,, the mass spectrometer selects only MS1-level analytes (usually by intensity) and fragments them sequentially. This provides good quality untargeted sample analysis, with quantification performed either at the MS1 level using label-free approaches or at the MS/MS level with isotopic labels, with the latter requiring additional sample preparation time and cost. With DDA, identification at the MS/MS level can be quite precise and specific given that the mass spectrometer is fragmenting only one analyte at a time. However, since DDA mode is stochastic and does not reproducibly select the same precursor ions for every run, it is not considered a particularly quantitative approach. Also, because high-intensity precursors are typically selected for MS/MS first, under-sampling the lower abundant analytes is often an issue in complex samples.

With DIA, the mass spectrometer does not require the initial detection of an MS peak to proceed to MS/MS analysis. Instead, it uses a wider Q1 isolation window and steps it across the entire m/z mass detection range, collecting MS/MS spectra on every detectable analyte that passes through each Q1 window. This means the full mass range is interrogated within an LC time frame, ensuring you get the full MS and MS/MS picture of every analyte that was detectable in your sample. This provides an unparalleled level of data completeness and allows quantification of MS1 and MS/MS level analytes in complex mixtures over a wide dynamic range. And since the instrument does not rely on intensity, which can vary between injections, determining which analyte has triggered missing data is not a problem with DIA. These complete digital maps of the samples can also be retrospectively analyzed when new information or new algorithms are available since all the data is already there.

There are many data independent analysis techniques available for customers these days. SWATH DIA reduces the risk of missing a critical component in your sample through an unbiased, comprehensive data acquisition strategy that delivers the data completeness your experiments demand.

SWATH DIA helps you acquire MS/MS data at incredibly fast acquisition rates with high sensitivity. This is key to getting the best DIA data because it allows you to run more Q1 isolation windows for higher specificity. It also helps ensure your cycle time is fast enough to maintain good LC peak sampling for reproducible and reliable peak integration and quantification quality, even with fast chromatography. Since our introduction of SWATH DIA so many years ago, we have continued to improve upon those initial capabilities. 

Being able to vary the Q1 isolation window size, allows you to use smaller Q1 m/z windows in the densest m/z region of your LC gradient. The result is increased specificity such that can capture deeper information on your sample, further reducing the risk of missing low abundance peaks in the most complex samples, while still maintaining broad mass range coverage to not miss anything.

Take proteomics as an example: many peptides lie in the 500-800 m/z mass range. With variable window SWATH DIA, you are able to shrink the Q1 window size in this mass range, delivering deeper coverage of peaks in this dense area of your sample, then use wider windows across the remaining mass range. This strategy maximizes the scan time of the instrument and gives you the power to dig deeper into the regions of greatest importance.

In the figure to the right, you can see how by adjusting the SWATH DIA window from a fixed 25 Da width at 80 msec each window (thus, 32 equal windows, above image), to 100 variable width windows at 25 msec each (below figure) delivers ~120% more proteins quantified in the sample. 

Zeno SWATH DIA, available exclusively on the ZenoTOF 7600 system, combines the reproducibility and precision of SWATH DIA with the significant gains in MS/MS sensitivity that the Zeno trap offers to deliver maximal information from each precious sample. Together, these powerful tools lead to a more comprehensive understanding of underlying biological changes.

The Zeno trap allows access to >90% duty cycles for all fragment ions, regardless of mass charge, or LC flow rate. This provides 4- to 20-fold sensitivity gains, all without compromising other important performance specifications such as resolution, mass accuracy and speed. The Zeno SWATH DIA workflow now capitalizes on these impressive MS/MS gains to enable widespread and comprehensive analyte coverage at new depths of detection and higher throughput than ever before.

For proteomics experiments, proteins across a broader and deeper range are available for detection, as shown in the figure to the right. At protein loads of <25 ng, we saw improvements of up to 2.5–3x more proteins quantified  (upper figure) and 4–5x more peptides quantified (lower figure) using Zeno SWATH DIA.

These gains allow scientists to run all kinds of experiments. You can delve into the potential of single-cell analysis, or run large-scale, high-throughput studies using robust high-flow chromatography, but with the luxury of consuming much less sample.

Additionally, the new “library-free” approach removes the initial effort involved in generating an experimental library and instead allows the creation of an in-silico library, making library creation truly “effort-free.”