In relative protein abundance determination from peptide intensities recorded in full mass scans, a major complication that affects quantitation accuracy is signal interference from coeluting ions of similar m/z values. Here, we present pQuant, a quantitation software tool that solves this problem. pQuant detects interference signals, identifies for each peptide a pair of least interfered isotopic chromatograms: one for the light and one for the heavy isotope-labeled peptide. On the basis of these isotopic pairs, pQuant calculates the relative heavy/light peptide ratios along with their 99.75% confidence intervals (CIs). From the peptides ratios and their CIs, pQuant estimates the protein ratios and associated CIs by kernel density estimation. We tested pQuant, Census and MaxQuant on data sets obtained from mixtures (at varying mixing ratios from 10:1 to 1:10) of light-and heavy-SILAC labeled HeLa cells or 14 N-and 15 N-labeled Escherichia coli cells. pQuant quantitated more peptides with better accuracy than Census and MaxQuant in all 14 data sets. On the SILAC data sets, the nonquantified "NaN" (not a number) ratios generated by Census, MaxQuant, and pQuant accounted for 2.5−10.7%, 1.8−2.7%, and 0.01−0.5% of all ratios, respectively. On the 14 N/ 15 N data sets, which cannot be quantified by MaxQuant, Census and pQuant produced 0.9−10.0% and 0.3−2.9% NaN ratios, respectively. Excluding these NaN results, the standard deviations of the numerical ratios calculated by Census or MaxQuant are 30−100% larger than those by pQuant. These results show that pQuant outperforms Census and MaxQuant in SILAC and 15 N-based quantitation.M uch progress has been made in mass spectrometry (MS)-based quantitative proteomics in recent years, as evidenced by numerous applications, such as biomarker discovery, 1 study of chromatin assembly and disassembly, 2 identification of insulin signaling targets, 3 and protein posttranslational modification (PTM). 4 Among the most commonly used quantitative strategies are full MS scan-based quantitation methods, such as SILAC (stable isotope labeling with amino acids in cell), 5 15 N-labeling, 6 and 18 O-labeling. 7 In these strategies, proteins are metabolically labeled with stable isotopes, digested into peptides, and then analyzed using liquid chromatography (LC)-MS/MS. Quantitation software tools are designed to extract the intensities of pairs of light (L, unlabeled) and heavy (H, labeled) peptides from full MS scans. The relative abundance ratio of a protein between two conditions is then calculated based on the ratios of its constituent peptides. 8 For high-complexity samples such as whole cell lysates, it is not uncommon that a peptide coelutes with another peptide or a nonpeptide contaminant of a similar m/z value. 9 The interference caused by coeluting ions of similar m/z values can seriously compromise the accuracy of quantitation. 10,11 We examined two leading quantitation software tools Census 12 and MaxQuant, 13 and found that a lot of the peptide quantitation results ...