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Robust optimization of SWATH-MS workflow for human blood serum proteome analysis using a quality by design approach

Lookup NU author(s): Dr Andrew Porter, Dr Dennis LendremORCiD, Professor John IsaacsORCiD



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


© 2021, The Author(s). Background: It is not enough to optimize proteomics assays. It is critical those assays are robust to operating conditions. Without robust assays, proteomic biomarkers are unlikely to translate readily into the clinic. This study outlines a structured approach to the identification of a robust operating window for proteomics assays and applies that method to Sequential Window Acquisition of all Theoretical Spectra Mass Spectroscopy (SWATH-MS). Methods: We used a sequential quality by design approach exploiting a fractional screening design to first identify critical SWATH-MS parameters, then using response surface methods to identify a robust operating window with good reproducibility, before validating those settings in a separate validation study. Results: The screening experiment identified two critical SWATH-MS parameters. We modelled the number of proteins and reproducibility as a function of those parameters identifying an operating window permitting robust maximization of the number of proteins quantified in human serum. In a separate validation study, these settings were shown to give good proteome-wide coverage and high quantification reproducibility. Conclusions: Using design of experiments permits identification of a robust operating window for SWATH-MS. The method gives a good understanding of proteomics assays and greater data-driven confidence in SWATH-MS performance.

Publication metadata

Author(s): Serrano-Blesa E, Porter A, Lendrem DW, Pitzalis C, Barton A, Treumann A, Isaacs JD

Publication type: Article

Publication status: Published

Journal: Clinical Proteomics

Year: 2021

Volume: 18

Issue: 1

Online publication date: 12/08/2021

Acceptance date: 21/07/2021

Date deposited: 26/08/2021

ISSN (print): 1542-6416

ISSN (electronic): 1559-0275

Publisher: BioMed Central Ltd


DOI: 10.1186/s12014-021-09323-z


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Funder referenceFunder name
MR/K015346/1Medical Research Council (MRC)