Data‐independent acquisition‐based SWATH ‐ MS for quantitative proteomics: a tutorial

Abstract

Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH‐MS is a specific variant of data‐independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH‐MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH‐MS data, a strategy based on peptide‐centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH‐MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH‐MS data using peptide‐centric scoring. Furthermore, concepts on how to improve SWATH‐MS data acquisition, potential trade‐offs of parameter settings and alternative data analysis strategies are discussed.

Partially overlapping sequential window acquisition of all theoretical mass spectra: A methodology to improve the spectra quality of veterinary drugs present at low concentrations in highly complex biological matrices

“…Figure is not only interesting from the analyte concentration point of view, but it also shows an advantage of the partially overlapping SWATH or SWATH techniques. As expected, the unit mass resolved MS (PRM) spectrum is cleaner than the partially overlapping SWATH spectrum. Yet, an intensive ion appearing within the partially overlapping SWATH spectrum, but absent within the PRM spectrum (at m/z 205), was discovered to be analyte‐ and not matrix‐related.…”

“…Hence it is the aim to detect all analyte‐related mass signals (zero missing detects) and avoid the recording of matrix‐related mass signals (zero false detects). Various attempts have been made to optimize these crucial issues . Concepts such as variable mass window SWATH (v‐SWATH) have been proposed.…”

A Review on Quantitative Multiplexed Proteomics

“…The Aebersold group introduced an approach, known as SWATH‐MS, to analyze these complex spectra, by using prior knowledge of the chromatographic and MS behavior of the peptides . This approach was recently reviewed by Ludwig et al., who described improvements to DIA and how SWATH‐MS could be used to analyze both total cell lysates and protein samples enriched for post‐translational modifications . The recent drastic improvements of DIA measurements are mostly due to computational advances .…”

Comparison of data acquisition modes with Orbitrap high‐resolution mass spectrometry for targeted and non‐targeted residue screening in aquacultured eel

“…In general, with DIA data collection, a limited group of precursor (MS1) ions are sequentially filtered through a quadrupole (with a wide isolation window) and then introduced into the collision cell to produce product (MS2) ions. DIA can also be performed with TOF instruments . DIA should theoretically provide increased sensitivity and selectivity along with better MS2 spectra than AIF or MS E for initial screening because a smaller number of precursor ions are being filtered and fragmented at any given time.…”