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In addition, the wide range of available normalization methods renders the choice of a suitable normalization method challenging. We systematically evaluated 17 normalization and 2 batch effect correction methods, originally developed for preprocessing DNA microarray data but widely applied in proteomics, on 6 publicly available spike-in and 3 label-free and tandem mass tag datasets. Opposed to state-of-the-art normalization practice, we found that a reduction in intragroup variation is not directly related to the effectiveness of the normalization methods. Furthermore, our results demonstrated that the methods RobNorm and Normics, specifically developed for proteomics data, in line with LoessF performed consistently well across the spike-in datasets, while EigenMS exhibited a high false-positive rate. Finally, based on experimental data, we show that normalization substantially impacts downstream analyses, and the impact is highly dataset-specific, emphasizing the importance of use-case-specific evaluations for novel proteomics datasets. For this, we developed the PROteomics Normalization Evaluator (PRONE), a unifying R package enabling comparative evaluation of normalization methods, including their impact on downstream analyses, while offering considerable flexibility, acknowledging the lack of universally accepted standards. PRONE is available on Bioconductor with a web application accessible at https:\/\/exbio.wzw.tum.de\/prone\/.<\/jats:p>","DOI":"10.1093\/bib\/bbaf201","type":"journal-article","created":{"date-parts":[[2025,4,21]],"date-time":"2025-04-21T07:33:18Z","timestamp":1745220798000},"source":"Crossref","is-referenced-by-count":5,"title":["Systematic evaluation of normalization approaches in tandem mass tag and label-free protein quantification data using PRONE"],"prefix":"10.1093","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7990-8385","authenticated-orcid":false,"given":"Lis","family":"Arend","sequence":"first","affiliation":[{"name":"Data Science in Systems Biology, TUM School of Life Sciences, Technical University of Munich , Maximus-von-Imhof Forum 3, 85354 Freising ,","place":["Germany"]},{"name":"Institute for Computational Systems Biology, University of Hamburg , Albert-Einstein-Ring 8-10, 22761 Hamburg 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1081\u00a0HV, Amsterdam ,","place":["The Netherlands"]}]},{"given":"Josch K","family":"Pauling","sequence":"additional","affiliation":[{"name":"LipiTUM, TUM School of Life Sciences, Technical University of Munich , Maximus-von-Imhof Forum 3, 85354 Freising ,","place":["Germany"]},{"name":"Institute for Clinical Chemistry and Laboratory Medicine, University Hospital and Faculty of Medicine Carl Gustav Carus of the Dresden University of Technology , Fetscherstr. 74, 01307 Dresden ,","place":["Germany"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6121-7105","authenticated-orcid":false,"given":"Stefan","family":"Kalkhof","sequence":"additional","affiliation":[{"name":"Department of Preclinical Development and Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI , Perlickstr. 1, 04103 Leipzig ,","place":["Germany"]},{"name":"Fraunhofer Cluster of Excellence Immune-Mediated Diseases CIMD , Perlickstr. 1, 04103 Leipzig ,","place":["Germany"]},{"name":"Institute for Bioanalysis, University of Applied Science Coburg , Friedrich-Streib-Str. 2, 96450 Coburg ,","place":["Germany"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0282-0462","authenticated-orcid":false,"given":"Jan","family":"Baumbach","sequence":"additional","affiliation":[{"name":"Institute for Computational Systems Biology, University of Hamburg , Albert-Einstein-Ring 8-10, 22761 Hamburg ,","place":["Germany"]},{"name":"Department of Mathematics and Computer Science, University of Southern Denmark , Campusvej 55, 5230 Odense ,","place":["Denmark"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0941-4168","authenticated-orcid":false,"given":"Markus","family":"List","sequence":"additional","affiliation":[{"name":"Data Science in Systems Biology, TUM School of Life Sciences, Technical University of Munich , Maximus-von-Imhof Forum 3, 85354 Freising ,","place":["Germany"]},{"name":"Munich Data Science Institute (MDSI), Technical University of Munich , Walther-von-Dyck-Stra\u00dfe 10, 85748 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