In data-independent acquisition strategies like SWATH acquisition, an expanded mass isolation window is stepped across a mass range covering the mass-to-charge (m/z) distribution of peptides and a full scan MS/MS spectrum is collected at each step. Post-acquisition, high resolution ion chromatograms are extracted for specific fragment ions of the peptides of interest, and these are integrated for quantitation. The information for extraction is stored in a spectral ion library and this contains all the proteins and peptides that you are interested in for the specific proteome you are studying.
There is a combination of acquisition attributes that need to come together to provide highest quality quantitative SWATH acquisition data.
- High resolution MS/MS is one key attribute as it enables target fragment ions to be found and extracted with good specificity from these complex MS/MS spectra. Typically for proteomics the high sensitivity MS/MS scan is used which provides 15-20K resolution.
- Cycle time is important as we want to have 6-8 points across the peak at half height for good quantitation / peak integration. So the MS/MS spectra must be acquired at high acquisition speed, we have investigated going as fast as 25 msec each (40Hz) and seen no decay in quantitation quality.
- Narrow Q1 isolation windows yield higher specificity in data analysis, which translates into better peak group detection and less possible interferences in quantitation. So high acquisition speed is important to take more, smaller steps to cover the full mass range. Using variable window acquisition really helps here to optimize the specificity / mass range tradeoff.
- Dynamic range is also a helpful attribute when analyzing proteomic samples that have a large range of protein abundance.
This is why the SCIEX QTOF, TripleTOF and ZenoTOF systems are so powerful for this acquisition technique; because high resolution MS/MS can be acquired at very high speeds, enabling much smaller isolation windows to be used.