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A method for independent estimation of false localisation rate for phosphoproteomics

View ORCID ProfileKerry A Ramsbottom, Ananth Prakash, Yasset Perez Riverol, Oscar Martin Camacho, Maria Martin, Juan Antonio Vizcaíno, Eric W Deutsch, Andrew R Jones
doi: https://doi.org/10.1101/2021.10.18.464791
Kerry A Ramsbottom
1Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, L69 3BX, United Kingdom
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  • ORCID record for Kerry A Ramsbottom
Ananth Prakash
2European Molecular Biology Laboratory, EMBL-European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridge, CB10 1SD, United Kingdom
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Yasset Perez Riverol
2European Molecular Biology Laboratory, EMBL-European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridge, CB10 1SD, United Kingdom
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Oscar Martin Camacho
1Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, L69 3BX, United Kingdom
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Maria Martin
2European Molecular Biology Laboratory, EMBL-European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridge, CB10 1SD, United Kingdom
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Juan Antonio Vizcaíno
2European Molecular Biology Laboratory, EMBL-European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridge, CB10 1SD, United Kingdom
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Eric W Deutsch
3Institute for Systems Biology, Seattle, Washington 98109, United States
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Andrew R Jones
1Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, L69 3BX, United Kingdom
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  • For correspondence: andrew.jones@liverpool.ac.uk
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Abstract

Phosphoproteomics methods are commonly employed in labs to identify and quantify the sites of phosphorylation on proteins. In recent years, various software tools have been developed, incorporating scores or statistics related to whether a given phosphosite has been correctly identified, or to estimate the global false localisation rate (FLR) within a given data set for all sites reported. These scores have generally been calibrated using synthetic data sets, and their statistical reliability on real datasets is largely unknown. As a result, there is considerable problem in the field of reporting incorrectly localised phosphosites, due to inadequate statistical control.

In this work, we develop the concept of using scoring and ranking modifications on a decoy amino acid, i.e. one that cannot be modified, to allow for independent estimation of global FLR. We test a variety of different amino acids to act as the decoy, on both synthetic and real data sets, demonstrating that the amino acid selection can make a substantial difference to the estimated global FLR. We conclude that while several different amino acids might be appropriate, the most reliable FLR results were achieved using alanine and leucine as decoys, although we have a preference for alanine due to the risk of potential confusion between leucine and isoleucine amino acids. We propose that the phosphoproteomics field should adopt the use of a decoy amino acid, so that there is better control of false reporting in the literature, and in public databases that re-distribute the data. Data are available via ProteomeXchange with identifier PXD028840.

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 4.0 International license.
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Posted October 19, 2021.
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A method for independent estimation of false localisation rate for phosphoproteomics
Kerry A Ramsbottom, Ananth Prakash, Yasset Perez Riverol, Oscar Martin Camacho, Maria Martin, Juan Antonio Vizcaíno, Eric W Deutsch, Andrew R Jones
bioRxiv 2021.10.18.464791; doi: https://doi.org/10.1101/2021.10.18.464791
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A method for independent estimation of false localisation rate for phosphoproteomics
Kerry A Ramsbottom, Ananth Prakash, Yasset Perez Riverol, Oscar Martin Camacho, Maria Martin, Juan Antonio Vizcaíno, Eric W Deutsch, Andrew R Jones
bioRxiv 2021.10.18.464791; doi: https://doi.org/10.1101/2021.10.18.464791

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