Improved data quality using variable Q1 window widths in SWATH acquisition

Data independent acquisition (DIA) on SCIEX ZenoTOF, TripleTOF and X-Series QTOF systems 

Introduction

In many of today’s mass spectrometry based workflows, the goal is to both identify and quantify a broad range of compounds in complex matrices. Whether it is quantifying differences in proteins or metabolites between complex biological samples, or screening for pesticides from a food matrix, the extreme complexity and dynamic range found in these matrices challenges traditional data dependent workflows to reproducibly and deeply interrogate the sample.

There has been a resurgence in the use of data independent acquisition (DIA) strategies to increase the reproducibility and comprehensiveness of data collection, enabled by recent technological innovations in both MS hardware and software. This acquisition strategy can now be routinely applied to complex sample matrices to collect high quality quantitative data. The most widely used of these techniques is SWATH acquisition on the SCIEX ZenoTOF, TripleTOF or QTOF systems^1,2.

In SWATH acquisition, Q1 isolation windows are stepped across the mass range in an LC timescale, transmitting populations of analytes for fragmentation, and high resolution composite MS/MS spectra are acquired at each step. The complexity of the MS/MS spectra and the resulting specificity depends on the number of analytes eluting off the column at the same time within the same m/z isolation window. The more narrow the Q1 isolation window, the better the signal/noise will be in the resulting data, but this must be balanced by how large a mass range needs to be interrogated (Figure 1).

The variable window SWATH acquisition strategy provides an elegant solution to this, by allowing the user to vary the size of the Q1 isolation window based on the density of analyte precursor masses (Figure 2). In this way, an optimal method can be easily built with a broad mass range for analyte coverage and with Q1 isolation windows for best specificity.

Key advantages of variable window SWATH acquisition

• Data independent acquisition using the SCIEX ZenoTOF, TripleTOF or QTOF systems using SWATH acquisition
  • High sensitivity, resolution and speed of MS/MS acquisition allows a large number of Q1 isolation windows with no loss in sensitivity or reproducibility
  • Up to 100 Q1 windows per cycle on TripleTOF 5600+ System, with upper mass limit 1250m/z
  • Up to 200 Q1 windows per cycle on the ZenoTOF, TripleTOF 6600+ and 6600 systems and X500 series instruments, with upper mass limit of 2250m/z
• Narrower Q1 isolation windows provide improved data quality through increased specificity
• SWATH Variable Window Calculator³ can be used to optimize the Q1 isolation window pattern for the matrix of interest, to achieve the right balance of analyte coverage and specificity
• Variable Window SWATH acquisition methods are easily built within the Analyst^® TF software or SCIEX OS software. 

Narrower isolation windows provide increased specificity

While many low level analyte detections in the SWATH acquisition data are good, some will have interferences in some of their MS/MS spectra and therefore their fragment ion XICs. In many cases, tighter Q1 windows can provide improved specificity by reducing the interferences from co-eluting analytes that have a similar m/z and produce similar mass fragment ions. An example of this improved signal/noise is shown in Figure 3 for a peptide from a low abundant protein in a complex digested proteome sample. 

As elements of the scoring depend on the consistency of spectral and/or chromatographic profiles of the fragments relative to a spectral ion library, it is clear how reduction of interferences can improve confident detection. It is often observed that significant gains in numbers of analytes quantified is achieved as the number of Q1 windows increases and window size decreases⁴. And of course, improved peak fidelity will greatly improve the consistency of peak integration and therefore quantitation quality.

Easy to create

The variable window SWATH acquisition method should be built based on the m/z distribution of analytes in the matrix of interest, therefore a single SWATH acquisition method can be built once and then used for that matrix over and over again, no matter what analytes are under investigation. The SWATH Variable Window Calculator³ can be used to compute the best Q1 window pattern for the matrix from the TOF MS m/z distribution of precursors. Then this information can be added to the method building software in Analyst TF software or SCIEX OS software to quickly build a final SWATH acquisition method (Figure 4).

Conclusions

The SWATH acquisition data independent acquisition strategy provides a comprehensive quantitative analysis of complex samples, providing full scan high resolution MS and MS/MS on all detectable compounds eluting off the column. These datasets can then be re-interrogated over and over again for compounds as new information emerges.

• SWATH acquisition can be optimized by balancing Q1 window width, the number of Q1 windows and accumulation time per window
  • Increases depth of quantitative coverage without compromising reproducibility due to improved S/N
• Optimizing window width based on m/z density provides further enhancement of specificity and data quality.
  • Allows for increased mass range to be covered
• SWATH Variable Window Calculator³ can be used to compute the window distribution for the complex sample of interest.

References

1. SWATH acquisition. SCIEX product page.
2. Gillet LC et al. (2012) Mol. Cell. Prot. 11(6), 1-17.
3. Download the SWATH Variable Window Calculator - Excel tool. 
4. Evolution of SWATH acquisition provides large gains in quantified proteins, SCIEX technical note RUO-MKT-02-5772-A.