Peptide filtering differently affects the performances of XIC-based quantification methods.

In bottom-up proteomics, data are acquired on peptides resulting from proteolysis. In XIC-based quantification, the quality of the estimation of protein abundance depends on how peptide data are filtered and on which quantification method is used to express peptide intensity as protein abundance. So far, these two questions have been addressed independently. Here, we studied to what extent the relative performances of the quantification methods depend on the filters applied to peptide intensity data. To this end, we performed a spike-in experiment using Universal Protein Standard to evaluate the performances of five quantification methods in five datasets obtained after application of four peptide filters. Estimated protein abundances were not equally affected by filters depending on the computation mode and the type of data for quantification. Furthermore, we found that filters could have contrasting effects depending on the quantification objective. Intensity modeling proved to be the most robust method, providing the best results in the absence of any filter. However, the different quantification methods can achieve similar performances when appropriate peptide filters are used. Altogether, our findings provide insights into how best to handle intensity data according to the quantification objective and the experimental design. SIGNIFICANCE: We believe that our results are of major importance because they address, as far as we know for the first time, the crossed-effects of peptide intensity data filtering and XIC-based quantification methods on protein quantification. While previous papers have dealt with peptide filtering independently of the quantification method, here we combined four peptide filters (based on peptide sharing between proteins, retention time variability, peptides occurrence and peptide intensity profiles) with five XIC-based quantification methods representing different modes of calculating protein abundances from peptide intensities. For these different combinations, we analyzed the quality of protein quantification in terms of precision, accuracy and linearity of response to increasing protein concentration using a spike-in experiment. We showed that not only filters effect on the estimation of protein abundances depend on the quantification methods but also that quantification methods can reach similar performances when appropriate peptide filters are used. Also, depending on the quantification objective, i.e. absolute or relative, filters can have contrasting effects and we demonstrated that protein quantification by the peptide intensity modeling was the most robust method.