| Abstract Title: | Investigation and Optimisation of Isolation Windows for SWATH Acquisition using Yeast |
| Presenter Name: | Ms Lisa Vuong |
| Co-authors: | Ms Aleksandra Chojnacka Ms Viktoriia Turkina Prof Saer Samanipour Prof Garry Corthals |
| Company/Organisation: | Van’t Hoff Institute for Molecular Sciences, University of Amsterdam |
| Country: | Netherlands |
Abstract Information :
Liquid chromatography coupled with tandem mass spectroscopy is commonly used for protein analysis with data-dependent acquisition (DDA) methods being the most preferred method due to its flexibility and simplicity of analysis. In DDA mode, the most abundant precursor ions are selected for fragmentation and comparison of the peptide fragments to a spectral library. The main issue with DDA methods is under-sampling as low abundant ions are not fragmented. Another acquisition method is data-independent acquisition, specifically, Sequential Window Acquisition of All Theoretical Mass Spectrometry (SWATH-MS). This acquisition method generates a fragment ion map of all the precursor ions within a defined precursor mass range, by stepping across the mass range at a defined window width. Utilising SWATH-MS allows for an increase in the number of identifications in comparison with DDA experiments but depending on the sample complexity, determining the correct SWATH-MS method to obtain high-quality spectra can be challenging due to several factors. This includes the type of isolation windows, i.e. variable or fixed, the width and the number of isolation windows. If too many windows are used, the quality of the spectra decreases with an increase in noise, and large windows can lead to co-fragmentation from different precursor ions in the same window. This study investigates and determines the optimal SWATH-MS method by analysis of yeast protein. Firstly, feature detection and componentisation are employed and to generate an experimental reference spectrum from DDA experiments, the degradation in the quality of the acquired spectra is quantified. This is done by assigning a quality score based on the fragments generated, relative intensities and the ratio of the product ions. Subsequently, the generation of an optimal reference spectrum by SWATH-MS is achieved by investigating the isolation window's type, number, and size. Lastly, the assessment of the quality of the acquired spectra from SWATH-MS is compared to the reference spectrum generated from DDA experiments which allows for an exploration into the ‘true’ number of windows that gives an optimal spectrum. By attaining this, it can increase the potential of using SWATH-MS for screening purposes.
