Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry

Abstract

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.

Complex‐centric proteome profiling by SEC ‐ SWATH ‐ MS

“…The top 20 mostintense precursors of charge state 2-5 were selected for CID fragmentation and MS2 spectra were collected for the range of 50-2,000 m/z, with 100 ms fill time cap and dynamic exclusion of precursor ions from reselection for 15 s, essentially as described (Collins et al, 2013). Data-independent acquisition (SWATH/DIA) mass spectrometry was performed using an updated scheme of 64 variably sized precursor co-isolation windows optimized for human cell lysate MS signal density (SWATHâ 2.0, essentially as described; Collins et al, 2017). SWATH cycles (64 × 50 ms accumulation time) were interspersed by MS1 survey scans for the range of 360-1,460 m/z with a 250 ms fill time cap, resulting in an overall period cycle time of 3,498 ms.…”

Systems proteomics approaches to study bacterial pathogens: application to Mycobacterium tuberculosis

“…For example, SWATH-MS, an implementation of the DIA strategy, combines the strengths of both aforementioned strategies, and can systematically and reproducibly analyze up of thousands proteins across various conditions and samples (Fig. 1d) [31,34]. To extract quantitative data from the highly complex and convoluted fragment ion spectra generated by DIA/SWATH-MS, prior knowledge about the fragmentation properties of specific peptides is typically employed [35].…”

Mass Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data

“…These concerns are particularly relevant for plasma proteomics studies where large-cohort projects are typically required to make biological conclusions, and minimizing technical noise is critical in identifying small, biologically significant changes. Towards this goal, researchers should consider incorporating broader technical performance characterization into their method development process, and should understand features such as multi-laboratory repeatability and identify common sources of confounding in their method of choice 103,104 . This information can help the plasma proteomics community move towards the ultimate goal of improved plasma characterization for applications in human health and biology.…”