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Comparison of TIMS-PASEF quantitative proteomics data-analysis workflows using FragPipe, DIA-NN, and Spectronaut from a user’s perspective

Alejandro Fernández-Vega, Federica Farabegoli, María M. Alonso-Martínez, View ORCID ProfileIgnacio Ortea
doi: https://doi.org/10.1101/2021.11.29.470373
Alejandro Fernández-Vega
1Clinical Proteomics Group, Instituto de Investigación e Innovación Biomédica de Cádiz (INiBICA), Cádiz 11009, Spain
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Federica Farabegoli
2ANFACO-CECOPESCA, Vigo 36310, Spain
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María M. Alonso-Martínez
2ANFACO-CECOPESCA, Vigo 36310, Spain
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Ignacio Ortea
1Clinical Proteomics Group, Instituto de Investigación e Innovación Biomédica de Cádiz (INiBICA), Cádiz 11009, Spain
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  • ORCID record for Ignacio Ortea
  • For correspondence: nacho.ortea@inibica.es
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Abstract

Data-independent acquisition (DIA) methods have gained great popularity in bottom-up quantitative proteomics, as they overcome the irreproducibility and under-sampling limitations of data-dependent acquisition (DDA). diaPASEF, recently developed for the timsTOF Pro mass spectrometers, has brought improvements to DIA, providing additional ion separation (in the ion mobility dimension) and increasing sensitivity. Several studies have benchmarked different workflows for DIA quantitative proteomics, but mostly using instruments from Sciex and Thermo, and therefore, the results are not extrapolable to diaPASEF data. In this work, using a real-life sample set like the one that can be found in any proteomics experiment, we compared the results of analyzing PASEF data with different combinations of library-based and library-free analysis, combining the tools of the FragPipe suite, DIA-NN and including MS1-level LFQ with DDA-PASEF data, and also comparing with the workflows possible in Spectronaut. We verified that library-independent workflows, not so efficient not so long ago, have greatly improved in the recent versions of the software tools, and now perform as well or even better than librarybased ones. We report here information so that the user who is going to conduct a relative quantitative proteomics study using a timsTOF Pro mass spectrometer can make an informed decision on how to acquire (diaPASEF for DIA analysis, or DDA-PASEF for MS1-level LFQ) the samples, and what can be expected depending on the data analysis tool used, among the different alternatives offered by the recently optimized tools for TIMS-PASEF data analysis.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted November 30, 2021.
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Comparison of TIMS-PASEF quantitative proteomics data-analysis workflows using FragPipe, DIA-NN, and Spectronaut from a user’s perspective
Alejandro Fernández-Vega, Federica Farabegoli, María M. Alonso-Martínez, Ignacio Ortea
bioRxiv 2021.11.29.470373; doi: https://doi.org/10.1101/2021.11.29.470373
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Comparison of TIMS-PASEF quantitative proteomics data-analysis workflows using FragPipe, DIA-NN, and Spectronaut from a user’s perspective
Alejandro Fernández-Vega, Federica Farabegoli, María M. Alonso-Martínez, Ignacio Ortea
bioRxiv 2021.11.29.470373; doi: https://doi.org/10.1101/2021.11.29.470373

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