WORLD PROTEOMICS COMES TO WESTMEAD!
Tips and tricks for building high quality spectral libraries for targeted data analysis of DIA datasets (Pre-Symposium Workshop)
1Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.
Targeted proteomics by selected reaction monitoring (SRM) or by SWATH MS data independent acquisition (DIA) relies on spectral reference libraries for peptide identification. Quality and coverage of these libraries are therefore of highest importance for the performance of the methods. In this workshop, we will present a set of advices to build high-quality assay libraries for optimal targeted proteomic analysis. Those include tips and tricks regarding the sample preparation, shotgun data acquisition, data conversion, data searching, generation of consensus spectra and compilation of the peptide MS coordinates into final assay lists. Altogether, those advices should help obtaining highest quality for the assays for optimal performance of the SWATH MS / DIA targeted data analysis.
New developments for highest reproducibility of SWATH MS analyses
Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.
SWATH MS is a LC-MS data independent acquisition method that records fragment ion maps for all analytes detectable in a given sample. Coupled with the targeted data extraction, the method can be considered as high-throughput, massively parallel targeted proteomic strategy that allows the query of any peptide/protein of interest in the acquired datasets.
In this presentation we will show new developments of the technology to attain highest reproducibility for the analyses. These developments include the implementation of the pressure cycling technology (PCT-SWATH) during the sample preparation for fast and highly reproducible lysis and digestion of cells or tissues. The reproducibility and consistency of the SWATH MS data acquisition and targeted protein quantification will be exemplified with the results of a large scale cross-laboratory benchmarking study. Finally, we will present new algorithms for multiple runs alignment strategy and for batch effect correction that have been implemented to improve even further the consistency of the quantification results across-runs. Altogether, the complete pipeline from sample preparation to data analysis achieves now highest levels of consistency and completeness for the final quantification matrix of the targeted proteins.