SWATH MS a data independent acquisition (DIA) method which aims to complement traditional mass spectrometry-based proteomics techniques such as shotgun and SRM methods. In essence, it allows a complete and permanent recording of all fragment ions of the detectable peptide precursors present in a biological sample. It thus combines the advantages of shotgun (high throughput) with those of SRM (high reproducibility and consistency).
The method can be best described in two steps: the data acquisition method and targeted data analysis approach building on the high-throughput SRM scoring mProphet approach developed in the Aebersold lab. In DIA acquisition set-up, the mass spectrometer steps within 2-4 seconds cycle time through a set of precursor acquisition windows designed to cover 400-1200 m/z as a whole mass range readily covered by a quadrupole mass analyzer and in which most of the tryptic peptide precursors of an organism fall. During each cycle, the mass spectrometer thus fragments all precursors from the quadrupole isolation window (e.g. 475 - 500 m/z for 25 Da wide windows) and records a complete, high accuracy fragment ion spectrum of all precursors selected in that isolation window. The same precursor isolation window is fragmented over and over at each cycle during the entire chromatographic separation, thus providing a time-resolved recording of the fragment ions of all the peptide precursors that elute on the chromatography. The SWATH MS data consists therefore of highly multiplexed fragment ion maps that are deterministically recorded over the user defined mass precursor mass range and chromatographic separation.
We are interested in the data-analysis challenge that is posed by DIA / SWATH MS data. Traditionally, DIA data have been analyzed by trying to reconstruct the lineage of precursor and fragment ions based on their chromatographic elution profile, and then analyzing the data using classical database searching strategies as in shotgun proteomics. However, these approaches may suffer from low sensitivity and propagation of errors due to the misassignment of fragment ions to precursor ions. In contrast, we have been actively developing methods and software for the automated targeted data analysis of SWATH MS datasets. The first generation of tools (OpenSWATH) extended the data analysis concept of SRM and offers now the researchers a tool set to achieve high-throughput DIA data extraction, scoring and statistical validation. We continue to develop novel algorithms to help the researchers to get more consistent and higher-quality analysis of their data and to obtain novel insights in the analysis of proteomes by exploring even deeper the SWATH MS data.