Comparative evaluation of label-free quantification methods for shotgun proteomics.

RATIONALE Label-free quantification (LFQ) is a popular strategy for shotgun proteomics. A variety of LFQ algorithms have been developed recently. However, a comprehensive comparison of the most commonly used LFQ methods is still rare, in part due to a lack of clear metrics for their evaluation and an annotated and quantitatively well-characterized data set. METHODS Five LFQ methods were compared: spectral counting based algorithms SIN , emPAI, and NSAF, and approaches relying on the extracted ion chromatogram (XIC) intensities, MaxLFQ and Quanti. We used three criteria for performance evaluation: coefficient of variation (CV) of protein abundances between replicates; analysis of variance (ANOVA); and the root-mean-square error of logarithmized calculated concentration ratios, referred to as standard quantification error (SQE). Comparison was performed using a quantitatively annotated publicly available data set. RESULTS The best results in terms of inter-replicate reproducibility were observed for MaxLFQ and NSAF, although they exhibited larger standard quantification errors. Using NSAF, all quantitatively annotated proteins were correctly identified in the Bonferronni-corrected results of the ANOVA test. SIN was found to be the most accurate in terms of SQE. Finally, the current implementations of XIC-based LFQ methods did not outperform the methods based on spectral counting for the data set used in this study. CONCLUSIONS Surprisingly, the performances of XIC-based approaches measured using three independent metrics were found to be comparable with more straightforward and simple MS/MS-based spectral counting approaches. The study revealed no clear leader among the latter. Copyright © 2017 John Wiley & Sons, Ltd.