Normalization methods in mass spectrometry-based analytical proteomics: A case study based on renal cell carcinoma datasets

Normalization is a crucial step in proteomics data analysis as it enables data adjustment and enhances comparability between datasets by minimizing multiple sources of variability, such as sampling, sample handling, storage, treatment, and mass spectrometry measurements. In this study, we investigated different normalization methods, including Z-score normalization, median divide normalization, and quantile normalization, to evaluate their performance using a case study based on renal cell carcinoma datasets. Our results demonstrate that when comparing datasets by pairs, both the Z-score and quantile normalization methods consistently provide better results in terms of the number of proteins identified and quantified as well as in identifying statistically significant up or down-regulated proteins. However, when three or more datasets are compared at the same time the differences are found to be negligible. [Display omitted] •Implications for advancing biomarker identification in renal carcinomas and analytical proteomics studies.•Comprehensive comparison of three normalization methods for proteomics datasets.•Rigorous analysis benchmarked against a validated method using immunohistochemistry.•Choice of normalization method loses criticality with more than two datasets.•Total protein approach demonstrated superior efficacy in biomarker discovery.