SWATH

SWATH (sequential window acquisition of all theoretical fragment ion spectra) is a new strategy for high throughput, label-free protein quantification. It generates global, quantitative protein maps using data-independent acquisition of collision-induced dissociation (CID) spectra of all precursor ions. As a data-independent acquisition (also referred to as MS/MSall) SWATH-MS has a greater coverage of peptide identification compared to classical discovery approaches.

Using known fingerprints of target peptides comprising precursor mass, chromatographic retention time and MRM transitions SWATH protein maps can be interrogated for targeted quantification of proteins of interest based on high resolution MRM-like signatures. SWATH acquires all MRM transitions of all precursors and thus does not require tedious assay development and allows for a more dynamic data interpretation compared to classical MRM experiments. New proteins can be added to the list of targets during the process of data interpretation without the requirement of additional data acquisition.
How does SWATH work? The mass spectrometer does not select and isolate a specific precursor ion for CID but fragments everything within a mass window of m/z 25 to acquire a single CID fragment-ion spectrum. To cover the full mass range between m/z 400-1250 the mass spectrometer sequentially acquires one full MS spectrum and about 34 CID-MS/MS spectra with isolation windows of m/z 25 during one cycle of roughly 3.5 seconds. Theoretically fragment ions of all precursor ions detectable throughout the selected mass range and along the chromatographic elution period are recorded. Such complex CID data however, cannot be matched to peptide sequences from databases through the commonly used search engines like Mascot, SEQUEST, ProteinPilot etc. Instead SWATH MS/MS data are searched against spectral libraries which can be generated from previous discovery data of data-dependent acquisitions.

SWATH (sequential window acquisition of all theoretical fragment ion spectra) is a new strategy for high throughput, label-free protein quantification. It generates global, quantitative protein maps using data-independent acquisition of collision-induced dissociation (CID) spectra of all precursor ions. As a data-independent acquisition (also referred to as MS/MSall) SWATH-MS has a greater coverage of peptide identification compared to classical discovery approaches.

Using known fingerprints of target peptides comprising precursor mass, chromatographic retention time and MRM transitions SWATH protein maps can be interrogated for targeted quantification of proteins of interest based on high resolution MRM-like signatures. SWATH acquires all MRM transitions of all precursors and thus does not require tedious assay development and allows for a more dynamic data interpretation compared to classical MRM experiments. New proteins can be added to the list of targets during the process of data interpretation without the requirement of additional data acquisition.

How does SWATH work? The mass spectrometer does not select and isolate a specific precursor ion for CID but fragments everything within a mass window of m/z 25 to acquire a single CID fragment-ion spectrum. To cover the full mass range between m/z 400-1250 the mass spectrometer sequentially acquires one full MS spectrum and about 34 CID-MS/MS spectra with isolation windows of m/z 25 during one cycle of roughly 3.5 seconds. Theoretically fragment ions of all precursor ions detectable throughout the selected mass range and along the chromatographic elution period are recorded. Such complex CID data however, cannot be matched to peptide sequences from databases through the commonly used search engines like Mascot, SEQUEST, ProteinPilot etc. Instead SWATH MS/MS data are searched against spectral libraries which can be generated from previous discovery data of data-dependent acquisitions.