Deconvolution of overlapping isotopic clusters improves quantification of stable isotope-labeled peptides

► When the mass shift between the labeled peptides is small, there can be the possibility for overlap of the isotopic clusters which will hamper quantification accuracy with a typical upwards bias for the heavier peptide. ► We investigated the impact of the overlapping peak issue with respect to dimethyl based quantification and we confirmed the need for correction. ► We present a tool that can correct overlapping issues when they arise which is based on modeling isotopic distributions. ► We demonstrate that our approach leads to improved accuracy and precision of protein quantification.